U.S. patent application number 16/318991 was filed with the patent office on 2019-10-31 for inhibitors of microbial beta-glucuronidase enzymes and uses thereof.
This patent application is currently assigned to The University of North Carolina at Chapel Hill. The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Ranathunga Arachchillage Yamu Ariyarathna, Stephen Frye, Lindsey James, Jian Jin, Sam Pellock, Matthew R. Redinbo.
Application Number | 20190330237 16/318991 |
Document ID | / |
Family ID | 60996088 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330237 |
Kind Code |
A1 |
Redinbo; Matthew R. ; et
al. |
October 31, 2019 |
INHIBITORS OF MICROBIAL BETA-GLUCURONIDASE ENZYMES AND USES
THEREOF
Abstract
Compounds and compositions are provided that comprise selective
b-glucuronidase inhibitors. The compounds and compositions can
ameliorate the side effects of chemotherapeutic agents and can
improve the efficacy of such agents, including irinotecan and
non-steroidal anti-inflammatory drugs.
Inventors: |
Redinbo; Matthew R.; (Chapel
Hill, NC) ; Jin; Jian; (New York, NY) ; James;
Lindsey; (Chapel Hill, NC) ; Pellock; Sam;
(Chapel Hill, NC) ; Ariyarathna; Ranathunga Arachchillage
Yamu; (Carrboro, NC) ; Frye; Stephen; (Chapel
Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Assignee: |
The University of North Carolina at
Chapel Hill
Chapel Hill
NC
|
Family ID: |
60996088 |
Appl. No.: |
16/318991 |
Filed: |
July 20, 2017 |
PCT Filed: |
July 20, 2017 |
PCT NO: |
PCT/US2017/043146 |
371 Date: |
January 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62365124 |
Jul 21, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 519/00 20130101;
C07D 495/14 20130101; A61P 1/12 20180101; C07D 513/14 20130101 |
International
Class: |
C07D 513/14 20060101
C07D513/14 |
Goverment Interests
GOVERNMENT SUPPORT
[0001] This invention was made with government support under Grant
CA098468 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A compound of Formula I: ##STR00038## wherein, R.sub.1 is
selected from the group consisting of: i. --OR.sub.A, wherein
R.sub.A is selected from the group consisting of H, CF.sub.3,
optionally substituted linear or branched C.sub.2-6 alkyl,
optionally substituted benzyl, and C.sub.3-8 cycloalkyl; ii.
--NR.sub.BR.sub.C, wherein R.sub.B and R.sub.C are each
independently selected from the group consisting of H, optionally
substituted benzyl, C.sub.2-5 heteroaryl, C.sub.2-5 heteroalkyl,
and C.sub.3-8 cycloalkyl; iii. C.sub.2-5 heteroaryl optionally
substituted with hydroxyl, halo, or amino; iv.
--N(CH.sub.2).sub.p--Z, wherein p is 1 or 2; and Z is heteroaryl or
heterocycloalkyl optionally substituted with hydroxyl, halo, or
amino; v. ##STR00039## wherein s is 1, 2, or 3; and R.sub.D is
amino, hydroxyl, halo, or linear or branched C.sub.1-6 alkyl; vi.
##STR00040## wherein R.sub.7 is H, linear or branched C.sub.1-6
alkyl optionally substituted with halo, amino, or hydroxyl; t is 1
or 2; q is 1 or 2, wherein R.sub.7 can form a bridge; and R.sub.8
is H, linear or branched C.sub.1-4 alkyl optionally substituted
with halo, hydroxyl, or amino; and vii. ##STR00041## wherein
R.sub.7 is H, linear or branched C.sub.1-6 alkyl optionally
substituted with halo, amino, or hydroxyl; t is 1 or 2; q is 1 or
2, wherein R.sub.7 can join to form a bridged compound; Y is a
counterion; and R.sub.9 is, independently in each instance, linear
or branched C.sub.1-4 alkyl optionally substituted with halo,
hydroxyl, or amino; R.sub.2 is aryl, heterocycloalkyl, or
--NR.sub.FR.sub.G, wherein; R.sub.F and R.sub.G are independently
selected from the group consisting of H, linear or branched
C.sub.1-6 alkyl optionally substituted with halo, amino or
hydroxyl, optionally substituted benzyl, and C.sub.3-8 cycloalkyl;
X is N or --CR.sub.3, wherein R.sub.3 is selected from H or
--NR.sub.IR.sub.J, wherein R.sub.I and R.sub.J are each
independently selected from H and linear or branched C.sub.1-6
alkyl, wherein said alkyl can be optionally substituted with
hydroxyl; and R.sub.4 and R.sub.5 are H or are taken together with
the carbon to which each is attached to form an optionally
substituted C.sub.5-7 membered ring; provided that when R.sub.4 and
R.sub.5 taken together form a 6-member unsubstituted ring, X is N
or CH, and R.sub.2 is morpholinyl, R.sub.1 is other than
piperazinyl or --NHCH.sub.2CH.sub.2NH.sub.2.
2. The compound of claim 1, wherein R.sub.4 and R.sub.5 are taken
together with the carbon to which each is attached to form an
optionally substituted C.sub.5-7 membered ring.
3. The compound of claim 2, wherein X is N.
4. The compound of claim 3, wherein R.sub.2 is
heterocycloalkyl.
5. (canceled)
6. (canceled)
7. The compound of claim 4, wherein R.sub.2 is morpholinyl.
8. The compound of claim 7, wherein R.sub.1 is --NR.sub.BR.sub.C,
wherein R.sub.B and R.sub.C are each independently selected from
the group consisting of H, optionally substituted benzyl, C.sub.2-5
heteroaryl, C.sub.2-5 heteroalkyl, and C.sub.3-5 cycloalkyl;
R.sub.1 is C.sub.2-5 heteroaryl optionally substituted with
hydroxyl, halo, or amino; or R.sub.1 is ##STR00042## wherein
R.sub.7 is H, linear or branched C.sub.1-6 alkyl optionally
substituted with halo, amino, or hydroxyl; t is 1 or 2; q is 1 or 2
and when q is 2 the two R.sub.7 groups may join to form a bridged
compound; and R.sub.8 is H, linear or branched C.sub.1-4 alkyl
optionally substituted with halo, hydroxyl, or amino.
9. (canceled)
10. The compound of claim 8, wherein R.sub.1 is ##STR00043##
wherein t and q are each 1, R.sub.8 is H, and R.sub.7 is
methyl.
11. (canceled)
12. (canceled)
13. The compound of claim 3, wherein R.sub.1 is ##STR00044##
wherein s is 1, 2, or 3; and R.sub.D is amino, hydroxyl, halo, or
linear or branched C.sub.1-6 alkyl; or R.sub.1 is ##STR00045##
wherein R.sub.7 is H, linear or branched C.sub.1-6 alkyl optionally
substituted with halo, amino, or hydroxyl; t is 1 or 2; q is 1 or
2, wherein R.sub.7 can join to form a bridged compound; and R.sub.8
is H, linear or branched C.sub.1-4 alkyl optionally substituted
with halo, hydroxyl, or amino.
14. (canceled)
15. The compound of claim 13, wherein R.sub.1 is ##STR00046##
wherein t and q are each 1, R.sub.7 is H, and R.sub.8 is H.
16. (canceled)
17. (canceled)
18. (canceled)
19. The compound of claim 15, wherein R.sub.2 is
heterocycloalkyl.
20. The compound of claim 19, which is ##STR00047##
21. The compound of claim 15, wherein R.sub.2 is --NR.sub.FR.sub.G,
wherein R.sub.F and R.sub.G are independently selected from the
group consisting of H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino or hydroxyl, optionally
substituted benzyl, and C.sub.3-8 cycloalkyl.
22. (canceled)
23. The compound of claim 21, which is selected from
##STR00048##
24. (canceled)
25. The compound of claim 1, wherein R.sub.4 and R.sub.5 are H.
26. The compound of claim 25, wherein R.sub.1 is ##STR00049##
wherein s is 1, 2, or 3; and R.sub.D is amino, hydroxyl, halo, or
linear or branched C.sub.1-6 alkyl; or R.sub.1 is ##STR00050##
wherein R.sub.7 is H, linear or branched C.sub.1-6 alkyl optionally
substituted with halo, amino, or hydroxyl; t is 1 or 2; q is 1 or
2, wherein R.sub.7 can join to form a bridged compound; and R.sub.8
is H, linear or branched C.sub.1-4 alkyl optionally substituted
with halo, hydroxyl, or amino.
27. (canceled)
28. (canceled)
29. (canceled)
30. The compound of claim 26, wherein R.sub.1 is piperazinyl.
31. The compound of claim 30, wherein R.sub.2 is H; aryl,
heterocycloalkyl, or --NR.sub.FR.sub.G, wherein R.sub.F and R.sub.G
are independently selected from the group consisting of H, linear
or branched C.sub.1-6 alkyl, optionally substituted benzyl, and
C.sub.3-8 cycloalkyl.
32. The compound of claim 31, wherein R.sub.2 is aryl.
33. (canceled)
34. The compound of claim 31, wherein R.sub.2 is
heterocycloalkyl.
35. (canceled)
36. (canceled)
37. (canceled)
38. The compound of claim 31, wherein R.sub.2 is --NR.sub.FR.sub.G,
wherein R.sub.F and R.sub.G are independently selected from the
group consisting of H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino or hydroxyl, optionally
substituted benzyl, and C.sub.3-8 cycloalkyl.
39. (canceled)
40. (canceled)
41. The compound of claim 38, selected from ##STR00051##
42. (canceled)
43. The compound of claim 1, having one of the following
structures: ##STR00052## ##STR00053## ##STR00054## ##STR00055##
##STR00056## ##STR00057## ##STR00058##
44. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier or excipient.
45. A method of ameliorating a side effect in a human in need
thereof, comprising administering to said human an effective amount
of a compound of claim 1.
Description
FIELD OF THE INVENTION
[0002] The invention relates generally to compounds which are
microbial .beta.-glucuronidase ("GUS")-specific inhibitors capable
of alleviating the toxicity of certain chemotherapeutic agents,
such as anticancer and non-steroidal anti-inflammatory drugs.
BACKGROUND
[0003] Irinotecan is a commonly used chemotherapeutic agent used in
the treatment of a variety of malignances. Unfortunately, patients
treated with Irinotecan often suffer side effects, such as severe
diarrhea, which increases patient suffering and thereby reduces the
viability for dose escalation and improved efficacy. Currently, no
effective therapy exists to overcome such diarrhea. It can be so
severe that it, in fact, becomes a dose limiting side effect.
[0004] The underlying mechanism of Irinotecan induced diarrhea has
been extensively investigated. The mechanism begins with bacteria
.beta.-glucuronidase (GUS) enzymes in the intestines converting the
nontoxic metabolite of Irinotecan, SN-38G, to toxic SN-38. The
compound SN-38 is the therapeutically active form of Irinotecan,
which when present in the intestines, leads to damage of intestinal
epithelial cells and diarrhea.
[0005] The microbial beta-glucuronidase (GUS) enzymes in the
mammalian gastrointestinal (GI) tract are the first established
drug targets in the microbiome. GUS enzymes have been shown to
reactivate drug metabolites to their toxic forms, which can
significantly damage the GI epithelium and reduce drug tolerance
and efficacy. (Wallace B. D., et al., Science, 330, 831-835
(2010)).
[0006] Compounds and methods that can safely and effectively
prevent the reactivation of drug metabolites are needed. The
subject matter described herein addresses this need.
BRIEF SUMMARY
[0007] In an embodiment, the subject matter described herein is
directed to compounds of Formula I, which are selective microbial
GUS inhibitors.
[0008] The compounds find use in inhibiting .beta.-glucuronidases
from hydrolyzing glucuronides, and thereby attenuate the side
effects seen in certain drugs, such as antineoplastic agents. In an
embodiment, the subject matter described herein is directed to
methods of treating a condition, such as gastrointestinal distress,
which often accompanies treatment of diseases, such as cancer, by
administering to a subject in need thereof a compound of Formula I
or a pharmaceutical formulation thereof.
[0009] Another embodiment includes pharmaceutical compositions
comprising a compound of Formula I or a pharmaceutically acceptable
salt and a pharmaceutically acceptable carrier.
[0010] Still another embodiment includes methods of inhibiting
.beta.-glucuronidases comprising contacting the
.beta.-glucuronidase with an effective amount of a compound of
Formula I or a pharmaceutically acceptable salt.
[0011] Still another embodiment includes methods of preparing the
compounds of Formula I.
[0012] Still further embodiments are as described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a 2.3 .ANG. resolution crystal structure of
EcGUS revealing that the di-piperizine compound 12 binds in two
orientations (A and B) at the GUS active site.
[0014] FIG. 2 shows a 2.4 .ANG. resolution crystal structure of a
microbial GUS in complex with compound 29 and a glucuronic acid
from a substrate molecule co-crystalized with enzyme and compound
29.
[0015] FIG. 3 shows a structural representation of an overlay of
the active sites of EcGUS (Blue), CpGUS, and SaGUS with Compound
29.
DETAILED DESCRIPTION
[0016] The selectively inhibiting agents of Formula I and
compositions thereof as disclosed herein can be used in connection
with methods for treating cancer and for reducing side effects of
antineoplastic agents, such as camptothecin-derived antineoplastic
agents. The gastrointestinal distress that typically accompanies
treatment with a particular chemotherapeutic agent can be
attenuated or ameliorated. The methods are also useful for
attenuating or improving any adverse reactions associated with
administration of glucuronidase-substrate agent(s) or compound(s).
The subject matter described herein includes compositions and
methods for inhibiting bacterial .beta.-glucuronidases and for
improving efficacy of camptothecin-derived antineoplastic agents or
glucuronidase-substrate agents or compounds by attenuating the
gastrointestinal distress caused by reactivation of glucuronidated
metabolites of such agents.
[0017] It has been found that inhibition of microbial
beta-glucuronidase (GUS) can alleviate the side effects and
toxicity of anticancer drugs, such as irinotecan, as well as
several non-steroidal anti-inflammatory drugs. Successful
inhibitors of microbial GUS enzymes have the following three
characteristics: potency, non-lethality to bacterial and mammalian
cells, and selectivity for the microbial GUS enzymes over the
mammalian GUS proteins. (Wallace, (2010)). As used herein,
"beta-glucuronidase," ".beta.-glucuronidase" and the like means an
enzyme (EC 3.2.1.31) capable of hydrolyzing .beta.-glucuronides,
but not .alpha.-glucuronides or .beta.-glucosides. (Basinska &
Florianczyk (2003) Ann. Univ. Mariae Curie Sklodowska Med.
58:386-389; Miles et al. (1955) J. Biol. Chem. 217:921-930). As
used herein, a "glucuronide" and the like means a substance
produced by linking glucuronic acid to another substance via a
glycosidic bond. Examples of glucuronides of interest herein
include, but are not limited to, glucuronides of
camptothecin-derived antineoplastic agents such as SN-38G
(7-ethyl-10-hydroxycamptothecin glucuronide). Further details of
microbiota beta-glucuronidase is disclosed in U.S. Pat. No.
8,557,808, herein incorporated by reference in its entirety.
[0018] Reactivation of inactive metabolites such as SN-38G to
active SN-38 occurs in the gastrointestinal tract and results from
bacterial .beta.-glucuronidases. The reactivated metabolites can
lead to a gastrointestinal distress such as diarrhea, which often
can be a dose-limiting side effect of the cancer therapy or the
therapy to treat any other conditions. As used herein,
"dose-limiting" indicates that the side effect from administration
of a camptothecin-derived antineoplastic agent or
glucuronidase-substrate agents or compounds prevents a subject in
need of cancer therapy or therapy to treat any other conditions
from receiving a recommended amount. As increasing amounts of the
camptothecin-derived antineoplastic agent or
glucuronidase-substrate agents or compounds are administered to a
subject, increased amounts of glucuronidated metabolites are
therefore available as a substrate for the bacterial
.beta.-glucuronidases. The resulting reactivated metabolites not
only adversely affect a subject's well-being by causing serious
side effects, particularly gastrointestinal distress, but also
impair treatment outcome by limiting the amount of the
camptothecin-derived antineoplastic agent or
glucuronidase-substrate agents or compounds that can be
administered to the subject.
[0019] While not being bound to theory, it is believed that the
compounds described herein are inhibitors of hydrolysis of a
covalent sugar-enzyme intermediate of the glycosyl hydrolase
reaction mechanism. As described herein, the compounds and methods
are selective for the bacterial microbiome that re-activates drugs
in the gut. Accordingly, the compounds and methods below are useful
for ameliorating the side effects of many drugs and treatments.
[0020] The presently disclosed subject matter will now be described
more fully hereinafter. However, many modifications and other
embodiments of the presently disclosed subject matter set forth
herein will come to mind to one skilled in the art to which the
presently disclosed subject matter pertains having the benefit of
the teachings presented in the foregoing descriptions. Therefore,
it is to be understood that the presently disclosed subject matter
is not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. In other words, the
subject matter described herein covers all alternatives,
modifications, and equivalents. In the event that one or more of
the incorporated literature, patents, and similar materials differs
from or contradicts this application, including but not limited to
defined terms, term usage, described techniques, or the like, this
application controls. Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in this field. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their
entirety.
I. Definitions
[0021] The term "substituent" refers to an atom or a group of atoms
that replaces a hydrogen atom on a molecule. The term "substituted"
denotes that a specified molecule bears one or more substituents.
The term "a compound of the formula" or "a compound of formula" or
"compounds of the formula" or "compounds of formula" refers to any
compound selected from the genus of compounds as defined by Formula
I.
[0022] As used herein, the term "alkyl" refers to a
straight-chained or branched hydrocarbon group. Examples of alkyl
groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, and
n-pentyl. Alkyl groups may be optionally substituted with one or
more substituents.
[0023] The term "benzyl" refers to a hydrocarbon with the formula
of C.sub.6H.sub.5CH.sub.2 where the point of attachment to the
group in question is at the CH.sub.2 position. The benzyl may be
substituted on the aromatic ring. In one embodiment, 0, 1, 2, 3, 4,
or 5 atoms of the aryl group may be substituted by a
substituent.
[0024] As used herein, the term "halogen", "hal" or "halo" means
--F, --Cl, --Br or --I.
[0025] The term "cycloalkyl" refers to a hydrocarbon 3-8 membered
monocyclic or 7-14 membered bicyclic ring system having at least
one saturated ring or having at least one non-aromatic ring,
wherein the non-aromatic ring may have some degree of unsaturation.
Cycloalkyl groups may be optionally substituted with one or more
substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each
ring of a cycloalkyl group may be substituted by a substituent.
Representative examples of cycloalkyl group include cyclopropyl,
cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl, cyclopentenyl,
cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
[0026] The term "aryl" refers to a hydrocarbon monocyclic, bicyclic
or tricyclic aromatic ring system. Aryl groups may be optionally
substituted with one or more substituents. In one embodiment, 0, 1,
2, 3, 4, 5 or 6 atoms of each ring of an aryl group may be
substituted by a substituent. Examples of aryl groups include
phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, and
the like.
[0027] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-4 ring heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, or S, and the remainder ring atoms
being carbon (with appropriate hydrogen atoms unless otherwise
indicated). Heteroaryl groups may be optionally substituted with
one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms
of each ring of a heteroaryl group may be substituted by a
substituent. Examples of heteroaryl groups include pyridyl,
furanyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl,
thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl,
pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl,
indazolyl, and the like.
[0028] The term "nitrogen-containing heteroaryl" refers to a
heteroaryl group having 1-4 ring nitrogen heteroatoms if
monocyclic, 1-6 ring nitrogen heteroatoms if bicyclic, or 1-9 ring
nitrogen heteroatoms if tricyclic.
[0029] The term "heterocycloalkyl" refers to a nonaromatic 3-8
membered monocyclic, 7-12 membered bicyclic, or 10-14 membered
tricyclic ring system comprising 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, S, B, P or Si, wherein the
nonaromatic ring system is completely saturated. Heterocycloalkyl
groups may be optionally substituted with one or more substituents.
In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a
heterocycloalkyl group may be substituted by a substituent.
Representative heterocycloalkyl groups include piperidinyl,
piperazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl,
1,3-dioxolanyl, tetrahydrofuryl, tetrahydrothienyl, thienyl, and
the like.
[0030] The term "tautomer" or "tautomeric form" refers to
structural isomers of different energies which are interconvertible
via a low energy barrier. For example, proton tautomers (also known
as prototropic tautomers) include interconversions via migration of
a proton, such as keto-enol and imine-enamine isomerizations.
Valence tautomers include interconversions by reorganization of
some of the bonding electrons.
[0031] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0032] The term "diastereomers" refers to stereoisomers with two or
more centers of dissymmetry and whose molecules are not minor
images of one another.
[0033] The term "enantiomers" refers to two stereoisomers of a
compound which are non-superimposable mirror images of one another.
An equimolar mixture of two enantiomers is called a "racemic
mixture" or a "racemate."
[0034] The term "isomers" or "stereoisomers" refers to compounds
which have identical chemical constitution, but differ with regard
to the arrangement of the atoms or groups in space.
[0035] Furthermore the compounds described herein may include
olefins having either geometry: "Z" refers to what is referred to
as a "cis" (same side) configuration whereas "E" refers to what is
referred to as a "trans" (opposite side) configuration.
[0036] A "solvate" refers to an association or complex of one or
more solvent molecules and a compound of the invention. Examples of
solvents that form solvates include, but are not limited to, water,
isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,
and ethanolamine. The term "hydrate" refers to the complex where
the solvent molecule is water.
[0037] A "metabolite" is a product produced through metabolism in
the body of camptothecin-derived antineoplastic agents or
glucuronidase-substrate agents or compounds. Metabolites may be
identified using routine techniques known in the art and their
activities determined using tests such as those described herein.
Such products may result, for example, from the oxidation,
hydroxylation, reduction, hydrolysis, amidation, deamidation,
esterification, deesterification, enzymatic cleavage, and the like,
of the administered compound.
[0038] The phrase "pharmaceutically acceptable" indicates that the
substance or composition must be compatible chemically and/or
toxicologically, with the other ingredients comprising a
formulation, and/or the subject being treated therewith.
[0039] The phrase "pharmaceutically acceptable salt" as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of a compound of the invention. Exemplary salts include, but
are not limited, to sulfate, citrate, acetate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal
(e.g., sodium and potassium) salts, alkaline earth metal (e.g.,
magnesium) salts, and ammonium salts. A pharmaceutically acceptable
salt may involve the inclusion of another molecule such as an
acetate ion, a succinate ion or other counter ion. The counter ion
may be any organic or inorganic moiety that stabilizes the charge
on the parent compound. Furthermore, a pharmaceutically acceptable
salt may have more than one charged atom in its structure.
Instances where multiple charged atoms are part of the
pharmaceutically acceptable salt, the salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counter ion. The
counter ions may have a positive or negative charge. A positive
counter ion may be mono- (e.g. an alkali metal or ammonium), di-
(e.g. an earth-alkali metal) or tri-valent (e.g. aluminium).
Non-limiting examples of alkali metal cations are Li.sup.+,
Na.sup.+, and K.sup.+. Non-limiting examples of negative counter
ions include chloride, bromide, iodide, sulfate, alkanesulfonate,
arylsulfonate, phosphate, perchlorate, tetrafluoroborate, and
nitrate.
[0040] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Non-limiting examples
of physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM.. In certain
embodiments, the pharmaceutically acceptable carrier is a
non-naturally occurring pharmaceutically acceptable carrier.
[0041] Use of the word "inhibitor" herein is meant to mean a
molecule that inhibits activity of microbial GUS enzymes. By
"inhibit" herein is meant to decrease the activity of the target
enzyme, as compared to the activity of that enzyme in the absence
of the inhibitor. In some embodiments, the term "inhibit" means a
decrease in GUS activity of at least about 5%, at least about 10%,
at least about 20%, at least about 25%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, or at least about 95%. In other embodiments, inhibit
means a decrease in GUS activity of about 5% to about 25%, about
25% to about 50%, about 50% to about 75%, or about 75% to 100%. In
some embodiments, inhibit means a decrease in GUS activity of about
95% to 100%, e.g., a decrease in activity of 95%, 96%, 97%, 98%,
99%, or 100%. Such decreases can be measured using a variety of
techniques that would be recognizable by one of skill in the art,
including in vitro assays.
[0042] As used herein, a "beta-glucoronidase inhibitor," "GUS
inhibitor" or an "inhibitor of GUS" is a compound that reduces,
inhibits, or otherwise diminishes one or more of the biological
activities of microbial GUS enzyme(s). Inhibition does not
necessarily indicate a total elimination of GUS activity. Instead,
the activity could decrease by a statistically significant amount
including, for example, a decrease of at least about 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 95% or 100% of the activity of GUS compared to an appropriate
control. In some embodiments, the compounds disclosed herein
reduce, inhibit, or otherwise diminish microbial GUS enzyme
activity.
[0043] By "specific inhibition" is intended an agent that reduces,
inhibits, or otherwise diminishes the activity of a specific
microbial GUS enzyme or ortholog greater than that of another
microbial GUS enzyme or ortholog. For example, a specific inhibitor
reduces at least one biological activity of a GUS ortholog, e.g.,
E. coli GUS (EcGUS) by an amount that is statistically greater than
the inhibitory effect of the compound on another GUS ortholog,
e.g., Streptococcus agalactiae (SaGUS) or Clostridium perfringens
(CpGUS). In some embodiments, the IC.sub.50 of the inhibitor for an
ortholog is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%,
1%, 0.1%, 0.01%, 0.001% or less of the IC.sub.50 of the inhibitor
for another ortholog. The presently disclosed compounds may or may
not be a specific inhibitor. A specific inhibitor reduces the
biological activity of a particular GUS by an amount that is
statistically greater than the inhibitory effect of the inhibitor
on any other GUS.
[0044] As used herein, "selectively inhibit" and the like means
that a .beta.-glucuronidase inhibitor reduces bacterial, but not
mammalian, .beta.-glucuronidase activity. That is, the
3-glucuronidase inhibitor can bind to and can prevent bacterial,
but not mammalian, 3-glucuronidases from hydrolyzing glucuronides.
Useful compounds of Formula I are selective for bacterial
.beta.-glucuronidase. That is, the compounds decrease activity of
the bacterial .beta.-glucuronidase by a statistically significant
amount as compared to the mammalian .beta.-glucuronidase.
Advantageously, the .beta.-glucuronidase inhibitors described
herein are selective for bacterial .beta.-glucuronidases. That is,
the compounds inhibit .beta.-glucuronidase in bacteria but do not
have inhibitory activity toward mammalian .beta.-glucuronidases,
including human .beta.-glucuronidase. While not intending to be
bound by any particular mechanism of action, the compounds appear
to bind a 12 residue loop in bacterial .beta.-glucuronidases that
hovers over an active site opening. The loop is not present in
mammalian .beta.-glucuronidases, which therefore can accommodate
larger substrates and cleave glucuronic acid moieties from
long-chain glycosaminoglycans. The .beta.-glucuronidase inhibitors
exhibit other advantages. For example, the compounds do not kill
the enteric bacteria or harm human epithelial cells, but are
effective against bacteria cultured under aerobic and anaerobic
conditions.
[0045] As used herein, "enteric bacteria" and the like mean the
normal bacteria that inhabit the human gastrointestinal track.
Examples of enteric bacteria include, but are not limited to,
Bacteroides sp. (e.g., Bacteroides vulgatus), Bifidobacterium sp.
(e.g., Bifidobacterium bifidum and Bifidobacterium infantis),
Catenabacterium sp., Clostridium sp., Corynebacterium sp.,
Enterococcus sp. (e.g., Enterococcus faecalis), Enterobacteriaceae
(e.g., Escherichia coli), Lactobacillus sp., Peptostreptococcus
sp., Propionibacterium sp., Proteus sp., Mycobacterium sp.,
Pseudomonas sp. (e.g., Pseudomonas aeruginosa), Staphylococcus sp.
(e.g., Staphylococcus epidermidis and Staphylococcus aureus) and
Streptococcus sp. (e.g., Streptococcus mitis). Because enteric
bacteria commensally inhabit the gastrointestinal tract, they
promote gastrointestinal health by preventing infection by
opportunistic bacteria like Clostridium difficle.
[0046] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder. The terms "alleviate" and
"attenuate" refer to the degree and rate of occurrence of the
objective and subjective symptomology associated with GI distress.
Beneficial or desired clinical results include, but are not limited
to, alleviation of symptoms, diminishment of extent of conditions,
stabilized (i.e., not worsening) condition, delay or slowing of
side effects, lessening of GI distress relative to the GI distress
suffered prior to receiving a compound of Formula I. "Treatment"
can also mean improved side effects as compared to expected side
effects if not receiving treatment. Those in need of treatment
include those already with the condition or disorder as well as
those prone to have the condition or disorder or those in which the
condition or disorder is to be prevented.
[0047] The term "administration" or "administering" includes routes
of introducing the compound(s) to a subject to perform their
intended function.
[0048] The term "effective amount" includes an amount effective, at
dosages and for periods of time necessary, to achieve the desired
result. An effective amount of compound may vary according to
factors such as the disease state, age, and weight of the subject,
and the ability of the compound to elicit a desired response in the
subject. Dosage regimens may be adjusted to provide the optimum
therapeutic response. An effective amount is also one in which any
toxic or detrimental effects (e.g., side effects) of the inhibitor
compound are outweighed by the therapeutically beneficial
effects.
[0049] The phrase "therapeutically effective amount" means an
amount of a compound of the present invention that (i) treats or
prevents the particular disease, condition, or disorder, (ii)
attenuates, ameliorates, or eliminates one or more symptoms of the
particular disease, condition, or disorder, or (iii) prevents or
delays the onset of one or more symptoms of the particular disease,
condition, or disorder described herein.
[0050] The term "subject" refers to animals such as mammals,
including, but not limited to, primates (e.g., humans), cows,
sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.
In certain embodiments, the subject is a human.
[0051] Additional definitions are also provided below.
II. Compounds
[0052] The compounds of Formula I, which also include
pharmaceutically acceptable salts, have the following
structure:
##STR00001##
wherein,
[0053] R.sub.1 is selected from the group consisting of: [0054] i.
--OR.sub.A, wherein R.sub.A is selected from the group consisting
of H, CF.sub.3, optionally substituted linear or branched C.sub.2-6
alkyl, optionally substituted benzyl, and C.sub.3-8 cycloalkyl;
[0055] ii. --NR.sub.BR.sub.C, wherein R.sub.B and R.sub.C are each
independently selected from the group consisting of H, linear or
branched C.sub.1-6 alkyl optionally substituted with amino,
optionally substituted benzyl, C.sub.2-5 heteroaryl, C.sub.2-5
heteroalkyl, and C.sub.3-8 cycloalkyl; [0056] iii. C.sub.2-5
heteroaryl optionally substituted with hydroxyl, halo, or amino;
[0057] iv. --N(CH.sub.2).sub.p--Z, wherein p is 1 or 2; and Z is
heteroaryl or heterocycloalkyl optionally substituted with
hydroxyl, halo, or amino; [0058] v.
[0058] ##STR00002## wherein s is 1, 2, or 3; and R.sub.D is amino,
hydroxyl, halo, or linear or branched C.sub.1-6 alkyl; and [0059]
vi.
[0059] ##STR00003## wherein [0060] R.sub.7 is H, linear or branched
C.sub.1-6 alkyl optionally substituted with halo, amino, or
hydroxyl; [0061] t is 1 or 2; [0062] q is 1 or 2, wherein R.sub.7
can form a bridge; and [0063] R.sub.8 is H, linear or branched
C.sub.1-4 alkyl optionally substituted with halo, hydroxyl, or
amino; or
[0064] R.sub.1 is selected from the group consisting of: [0065] i.
--OR.sub.A, wherein R.sub.A is selected from the group consisting
of H, CF.sub.3, optionally substituted linear or branched C.sub.2-6
alkyl, optionally substituted benzyl, and C.sub.3-8 cycloalkyl;
[0066] ii. --NR.sub.BR.sub.C, wherein R.sub.B and R.sub.C are each
independently selected from the group consisting of H, linear or
branched C.sub.1-6 alkyl optionally substituted with amino,
optionally substituted benzyl, C.sub.2-5 heteroaryl, C.sub.2_.sub.5
heteroalkyl, and C.sub.3-8 cycloalkyl; [0067] iii. C.sub.2-5
heteroaryl optionally substituted with hydroxyl, halo, or amino;
[0068] iv. --N(CH.sub.2).sub.p--Z, wherein p is 1 or 2; and Z is
heteroaryl or heterocycloalkyl optionally substituted with
hydroxyl, halo, or amino; [0069] v.
[0069] ##STR00004## wherein s is 1, 2, or 3; and R.sub.D is amino,
hydroxyl, halo, or linear or branched C.sub.1-6 alkyl; [0070]
vi.
[0070] ##STR00005## wherein [0071] R.sub.7 is H, linear or branched
C.sub.1-6 alkyl optionally substituted with halo, amino, or
hydroxyl; [0072] t is 1 or 2; [0073] q is 1 or 2, wherein R.sub.7
can form a bridge; and [0074] R.sub.8 is H, linear or branched
C.sub.1-4 alkyl optionally substituted with halo, hydroxyl, or
amino; and [0075] vii.
[0075] ##STR00006## wherein [0076] R.sub.7 is H, linear or branched
C.sub.1-6 alkyl optionally substituted with halo, amino, or
hydroxyl; [0077] t is 1 or 2; [0078] q is 1 or 2, wherein R.sub.7
can form a bridge; [0079] Y is a counterion; and [0080] R.sub.9 is,
independently in each instance, linear or branched C.sub.1-4 alkyl
optionally substituted with halo, hydroxyl, or amino;
[0081] R.sub.2 is H, aryl, heterocycloalkyl, or --NR.sub.FR.sub.G,
wherein [0082] R.sub.F and R.sub.G are independently selected from
the group consisting of H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino or hydroxyl, optionally
substituted benzyl, and C.sub.3-8 cycloalkyl;
[0083] X is N or --CR.sub.3, wherein [0084] R.sub.3 is selected
from the group consisting of H, halogen, --OR.sub.I, and
--NR.sub.IR.sub.J, wherein [0085] R.sub.I and R.sub.J are each
independently selected from H and linear or branched C.sub.1-6
alkyl, wherein said alkyl can be optionally substituted with
hydroxyl; and
[0086] R.sub.4 and R.sub.5 are H or are taken together with the
carbon to which each is attached to form an optionally substituted
C.sub.5-7 membered ring;
[0087] provided that when R.sub.4 and R.sub.5 taken together form a
6-member unsubstituted ring, X is N or CH, and R.sub.2 is
morpholinyl, R.sub.1 is other than piperazinyl or
--NHCH.sub.2CH.sub.2NH.sub.2.
[0088] In embodiments, R.sub.4 and R.sub.5 are taken together with
the carbon to which each is attached to form an optionally
substituted C.sub.5-7 membered ring.
[0089] In any above embodiment, X is N.
[0090] In any above embodiment, R.sub.2 is heterocycloalkyl.
[0091] In any above embodiment, R.sub.2 is a 4-8 membered ring
containing 1 or 2 nitrogen atoms.
[0092] In any above embodiment, R.sub.2 is chosen from the group
consisting of:
##STR00007##
[0093] In any above embodiment, R.sub.2 is morpholinyl.
[0094] In any above embodiment, R.sub.1 is [0095]
--NR.sub.BR.sub.C, wherein R.sub.B and R.sub.C are each
independently selected from the group consisting of H, linear or
branched C.sub.1-6 alkyl optionally substituted with amino,
optionally substituted benzyl, C.sub.2-5 heteroaryl, C.sub.2-5
heteroalkyl, and C.sub.3-8 cycloalkyl; [0096] R.sub.1 is C.sub.2-5
heteroaryl optionally substituted with hydroxyl, halo, or amino;
or, [0097] R.sub.1 is
[0097] ##STR00008## wherein [0098] R.sub.7 is H, linear or branched
C.sub.1-6 alkyl optionally substituted with halo, amino, or
hydroxyl; [0099] t is 1 or 2; [0100] q is 1 or 2 and when q is 2
the two R.sub.7 groups may join to form a bridged compound; and
[0101] R.sub.8 is H, linear or branched C.sub.1-4 alkyl optionally
substituted with halo, hydroxyl, or amino.
[0102] In any above embodiment, R.sub.1 is
##STR00009##
wherein R.sub.7 is H, linear or branched C.sub.1-6 alkyl optionally
substituted with halo, amino, or hydroxyl; [0103] t is 1 or 2;
[0104] q is 1 or 2, wherein R.sub.7 can join to form a bridged
compound; and [0105] R.sub.8 is H, linear or branched C.sub.1-4
alkyl optionally substituted with halo, hydroxyl, or amino.
[0106] In any above embodiment, t and q are each 1.
[0107] In any above embodiment, R.sub.8 is H.
[0108] In any above embodiment, R.sub.7 is methyl.
[0109] In any above embodiment, R.sub.1 is
##STR00010##
wherein [0110] s is 1, 2, or 3; and [0111] R.sub.D is amino,
hydroxyl, halo, or linear or branched C.sub.1-6 alkyl; or
[0112] R.sub.1 is
##STR00011##
wherein [0113] R.sub.7 is H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino, or hydroxyl; [0114] t is 1
or 2; [0115] q is 1 or 2, wherein R.sub.7 can join to form a
bridged compound; and [0116] R.sub.8 is H, linear or branched
C.sub.1-4 alkyl optionally substituted with halo, hydroxyl, or
amino.
[0117] In any above embodiment, R.sub.1 is particularly
##STR00012##
wherein [0118] R.sub.7 is H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino, or hydroxyl; [0119] t is 1
or 2; [0120] q is 1 or 2, wherein R.sub.7 can join to form a
bridged compound; and [0121] R.sub.8 is H, linear or branched
C.sub.1-4 alkyl optionally substituted with halo, hydroxyl, or
amino.
[0122] In any above embodiment, t and q are each 1.
[0123] In any above embodiment, R.sub.7 is H.
[0124] In any above embodiment, R.sub.8 is H.
[0125] In any above embodiment, R.sub.2 is H, heterocycloalkyl, or
--NR.sub.FR.sub.G, wherein [0126] R.sub.F and R.sub.G are
independently selected from the group consisting of H, linear or
branched C.sub.1-6 alkyl optionally substituted with halo, amino or
hydroxyl, optionally substituted benzyl, and C.sub.3-8
cycloalkyl.
[0127] In any above embodiment, R.sub.2 is heterocycloalkyl.
[0128] In a particular embodiment, the compound of Formula I is
##STR00013##
[0129] In any above embodiment, R.sub.2 is --NR.sub.FR.sub.G,
wherein [0130] R.sub.F and R.sub.G are independently selected from
the group consisting of H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino or hydroxyl, optionally
substituted benzyl, and C.sub.3-8 cycloalkyl.
[0131] In any above embodiment, R.sub.F and R.sub.G are
independently selected from H or linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino or hydroxyl.
[0132] In a particular embodiment, the compound of Formula I is
##STR00014##
[0133] In any above embodiment, wherein R.sub.4 and R.sub.5 are
H.
[0134] In any above embodiment, R.sub.1 is
##STR00015##
wherein [0135] s is 1, 2, or 3; and [0136] R.sub.D is amino,
hydroxyl, halo, or linear or branched C.sub.1-6 alkyl; or
[0137] R.sub.1 is
##STR00016##
wherein [0138] R.sub.7 is H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino, or hydroxyl; [0139] t is 1
or 2; [0140] q is 1 or 2, wherein R.sub.7 can join to form a
bridged compound; and [0141] R.sub.8 is H, linear or branched
C.sub.1-4 alkyl optionally substituted with halo, hydroxyl, or
amino.
[0142] In any above embodiment, R.sub.1 is
##STR00017##
wherein [0143] R.sub.7 is H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino, or hydroxyl; [0144] t is 1
or 2; [0145] q is 1 or 2, wherein R.sub.7 can join to form a
bridged compound; and [0146] R.sub.8 is H, linear or branched
C.sub.1-4 alkyl optionally substituted with halo, hydroxyl, or
amino.
[0147] In any above embodiment, t and q are each 1.
[0148] In any above embodiment, R.sub.7 is H.
[0149] In any above embodiment, R.sub.1 is piperazinyl.
[0150] In any above embodiment, R.sub.2 is H, aryl,
heterocycloalkyl, or --NR.sub.FR.sub.G, wherein [0151] R.sub.F and
R.sub.G are independently selected from the group consisting of H,
linear or branched C.sub.1-6 alkyl, optionally substituted benzyl,
and C.sub.3-8 cycloalkyl.
[0152] In any above embodiment, R.sub.2 is aryl.
[0153] In any above embodiment, R.sub.2 is phenyl.
[0154] In any above embodiment, R.sub.2 is heterocycloalkyl.
[0155] In any above embodiment, R.sub.2 is a 4-8 membered ring
containing 1 or 2 nitrogen atoms contained within the ring.
[0156] In any above embodiment, R.sub.2 is chosen from the group
consisting of:
##STR00018##
[0157] In any above embodiment, R.sub.2 is morpholinyl.
[0158] In any above embodiment, R.sub.2 is --NR.sub.FR.sub.G,
wherein [0159] R.sub.F and R.sub.G are independently selected from
the group consisting of H, linear or branched C.sub.1-6 alkyl
optionally substituted with halo, amino or hydroxyl, optionally
substituted benzyl, and C.sub.3-8 cycloalkyl.
[0160] In any above embodiment, R.sub.F and R.sub.G are
independently H or linear or branched C.sub.1-6 alkyl optionally
substituted with halo, amino or hydroxyl.
[0161] In any above embodiment, R.sub.F and R.sub.G are
independently H or unsubstituted linear C.sub.1-6 alkyl.
[0162] In a particular embodiment, the compound of Formula I is
or,
##STR00019##
[0163] In embodiments, the compound of Formula I is selected from
the group consisting of:
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026##
[0164] In embodiments, the subject matter described herein is
directed to a pharmaceutical composition comprising a compound of
Formula I and a pharmaceutically acceptable carrier or
excipient.
[0165] In embodiments, the subject matter described herein is
directed to a method of treating a disease in a subject in need
thereof, comprising administering to the subject an effective
amount of a compound of Formula I.
[0166] The subject matter described herein also includes
pharmaceutically acceptable salts of compounds of Formula I.
[0167] In any embodiment above, the compound of Formula I
specifically inhibits one microbial GUS enzyme or ortholog greater
than that of another microbial GUS enzyme or ortholog. In an
embodiment, the IC.sub.50 of the inhibitor for an ortholog is about
90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%,
0.001% or less of the IC.sub.50 of the inhibitor for another
ortholog. In an embodiment, the compound of Formula I specifically
inhibits EcGUS in comparison to other GUS orthologs. In an
embodiment, the compound of Formula I specifically inhibits SaGUS
in comparison to other GUS orthologs. In an embodiment, the
compound of Formula I specifically inhibits CpGUS in comparison to
other GUS orthologs.
[0168] In a further embodiment, the compound of Formula I has an
EcGUS IC.sub.50 in a range from about 1% to about 10% the IC.sub.50
of any other GUS ortholog. In further embodiments, the compound of
Formula I has a EcGUS IC.sub.50 in a range from about 10% to about
25%, about 25% to about 50%, or about 50% to about 90% the
IC.sub.50 of any other GUS ortholog. In a further embodiment, the
compound of Formula I has a SaGUS IC.sub.50 in a range from about
20% to about 30% the IC.sub.50 of any other GUS ortholog. In
further embodiments, the compound of Formula I has a SaGUS
IC.sub.50 in a range from about 30% to about 50%, about 50% to
about 75%, or about 75% to about 90% the IC.sub.50 of any other GUS
ortholog.
[0169] If the compound of Formula I is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of the free
base with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric
acid and the like, or with an organic acid, such as acetic acid,
maleic acid, succinic acid, mandelic acid, fumaric acid, malonic
acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a
pyranosidyl acid, such as glucuronic acid or galacturonic acid, an
alpha hydroxy acid, such as citric acid or tartaric acid, an amino
acid, such as aspartic acid or glutamic acid, an aromatic acid,
such as benzoic acid or cinnamic acid, a sulfonic acid, such as
p-toluenesulfonic acid or ethanesulfonic acid, or the like.
[0170] Illustrative examples of other suitable salts include, but
are not limited to, organic salts derived from amino acids, such as
glycine and arginine, ammonia, primary, secondary, and tertiary
amines, and cyclic amines, such as piperidine, morpholine and
piperazine, and inorganic salts derived from sodium, calcium,
potassium, magnesium, manganese, iron, copper, zinc, aluminum and
lithium.
III. Compositions
[0171] Compounds of Formula I can be formulated in accordance with
standard pharmaceutical practice as a pharmaceutical composition.
According to this aspect, there is provided a pharmaceutical
composition comprising a compound of Formula I in association with
a pharmaceutically acceptable excipient, such as a carrier or
diluent.
[0172] A typical formulation is prepared by mixing a Formula I
compound and one or more excipients. Suitable excipients are well
known to those skilled in the art and include materials such as
carbohydrates, waxes, water soluble and/or swellable polymers,
hydrophilic or hydrophobic materials, gelatin, oils, solvents,
water and the like. The particular excipients used will depend upon
the means and purpose for which the compound of Formula I is being
applied. Solvents are generally selected based on solvents
recognized by persons skilled in the art as safe (GRAS) to be
administered to a mammal. In general, safe solvents are non-toxic
aqueous solvents such as water and other non-toxic solvents that
are soluble or miscible in water. Suitable aqueous solvents include
water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG
400, PEG 300), etc. and mixtures thereof. The formulations may also
include one or more buffers, stabilizing agents, surfactants,
wetting agents, lubricating agents, emulsifiers, suspending agents,
preservatives, antioxidants, opaquing agents, glidants, processing
aids, colorants, sweeteners, perfuming agents, flavoring agents and
other known additives to provide an elegant presentation of the
drug (i.e., a compound of Formula I or pharmaceutical composition
thereof) or aid in the manufacturing of the pharmaceutical product
(i.e., medicament).
[0173] The formulations may be prepared using conventional
dissolution and mixing procedures. For example, the bulk drug
substance (i.e., compound of Formula I or stabilized form of the
Formula I compound) (e.g., complex with a cyclodextrin derivative
or other known complexation agent) is dissolved in a suitable
solvent in the presence of one or more of the excipients described
above. The compound of Formula I is typically formulated into
pharmaceutical dosage forms to provide an easily controllable
dosage of the drug and to enable patient compliance with the
prescribed regimen.
[0174] The pharmaceutical composition (or formulation) for
application may be packaged in a variety of ways depending upon the
method used for administering the drug. Generally, an article for
distribution includes a container having deposited therein the
pharmaceutical formulation in an appropriate form. Suitable
containers are well known to those skilled in the art and include
materials such as bottles (plastic and glass), sachets, ampoules,
plastic bags, metal cylinders, and the like. The container may also
include a tamper-proof assemblage to prevent indiscreet access to
the contents of the package. In addition, the container has
deposited thereon a label that describes the contents of the
container. The label may also include appropriate warnings. In one
embodiment, the container is a blister pack.
[0175] Pharmaceutical formulations may be prepared for various
routes and types of administration. For example, a compound of
Formula I having the desired degree of purity may optionally be
mixed with pharmaceutically acceptable excipients (Remington's
Pharmaceutical Sciences (1980) 16.sup.th edition, Osol, A. Ed.,
Mack Publishing Co., Easton, Pa.), in the form of a lyophilized
formulation, milled powder, or an aqueous solution. Formulation may
be conducted by mixing at ambient temperature at the appropriate
pH, and at the desired degree of purity, with physiologically
acceptable carriers, i.e., carriers that are non-toxic to
recipients at the dosages and concentrations employed. The pH of
the formulation depends mainly on the particular use and the
concentration of compound, but may range from about 3 to about 8.
Formulation in an acetate buffer at pH 5 is a suitable
embodiment.
[0176] The compounds of Formula I can be sterile. In particular,
formulations to be used for in vivo administration must be sterile.
Such sterilization is readily accomplished by filtration through
sterile filtration membranes.
[0177] The compound ordinarily can be stored as a solid
composition, a lyophilized formulation or as an aqueous
solution.
[0178] The pharmaceutical compositions comprising a compound of
Formula I can be formulated, dosed and administered in a fashion,
i.e., amounts, concentrations, schedules, course, vehicles and
route of administration, consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The "therapeutically effective
amount" of the compound to be administered will be governed by such
considerations. Acceptable excipients are nontoxic to recipients at
the dosages and concentrations employed, and may include buffers
such as phosphate, citrate and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
polysorbates (e.g., TWEEN.TM.), poloxamers (e.g., PLURONICS.TM.) or
polyethylene glycol (PEG). The active pharmaceutical ingredients
may also be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences.
[0179] Sustained-release preparations of Formula I compounds may be
prepared. Suitable examples of sustained-release preparations
include semipermeable matrices of solid hydrophobic polymers
containing a compound of Formula I, which matrices are in the form
of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl
alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of
L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable
ethylene-vinyl acetate, degradable lactic acid-glycolic acid
copolymers such as injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate (LUPRON
DEPOT.TM.) and poly-D-(-)-3-hydroxybutyric acid.
[0180] The formulations include those suitable for the
administration routes detailed herein. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art of pharmacy. Techniques
and formulations generally are found in Remington's Pharmaceutical
Sciences. Such methods include the step of bringing into
association the active ingredient with the carrier which
constitutes one or more accessory ingredients. In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0181] Formulations of a compound of Formula I suitable for oral
administration may be prepared as discrete units such as pills,
capsules, cachets or tablets each containing a predetermined amount
of a compound of Formula I.
[0182] Compressed tablets may be prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such
as a powder or granules, optionally mixed with a binder, lubricant,
inert diluent, preservative, surface active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered active ingredient moistened with an inert
liquid diluent. The tablets may optionally be coated or scored and
optionally are formulated so as to provide slow or controlled
release of the active ingredient therefrom.
[0183] Tablets, troches, lozenges, aqueous or oil suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
e.g., gelatin capsules, syrups or elixirs may be prepared for oral
use. Formulations of compounds of Formula I intended for oral use
may be prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
may contain one or more agents including sweetening agents,
flavoring agents, coloring agents and preserving agents, in order
to provide a palatable preparation. Tablets containing the active
ingredient in admixture with non-toxic pharmaceutically acceptable
excipient which are suitable for manufacture of tablets are
acceptable. These excipients may be, for example, inert diluents,
such as calcium or sodium carbonate, lactose, calcium or sodium
phosphate; granulating and disintegrating agents, such as maize
starch, or alginic acid; binding agents, such as starch, gelatin or
acacia; and lubricating agents, such as magnesium stearate, stearic
acid or talc. Tablets may be uncoated or may be coated by known
techniques including microencapsulation to delay disintegration and
adsorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate alone
or with a wax may be employed.
[0184] Aqueous suspensions of Formula I compounds contain the
active materials in admixture with excipients suitable for the
manufacture of aqueous suspensions. Such excipients include a
suspending agent, such as sodium carboxymethylcellulose,
croscarmellose, povidone, methylcellulose, hydroxypropyl
methylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth and gum acacia, and dispersing or wetting agents such as
a naturally occurring phosphatide (e.g., lecithin), a condensation
product of an alkylene oxide with a fatty acid (e.g.,
polyoxyethylene stearate), a condensation product of ethylene oxide
with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0185] The pharmaceutical compositions of compounds of Formula I
may be in the form of a sterile injectable preparation, such as a
sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, such 1,3-butanediol.
The sterile injectable preparation may also be prepared as a
lyophilized powder. Among the acceptable vehicles and solvents that
may be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile fixed oils may
conventionally be employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid may likewise be used in the preparation of
injectables.
[0186] The amount of active ingredient that may be combined with
the carrier material to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a time-release formulation intended
for oral administration to humans may contain approximately 1 to
1000 mg of active material compounded with an appropriate and
convenient amount of carrier material which may vary from about 5
to about 95% of the total compositions (weight:weight). The
pharmaceutical composition can be prepared to provide easily
measurable amounts for administration. For example, an aqueous
solution intended for intravenous infusion may contain from about 3
to 500 .mu.g of the active ingredient per milliliter of solution in
order that infusion of a suitable volume at a rate of about 30
mL/hr can occur.
[0187] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0188] Formulations suitable for topical administration to the eye
also include eye drops wherein the active ingredient is dissolved
or suspended in a suitable carrier, especially an aqueous solvent
for the active ingredient. The active ingredient is preferably
present in such formulations in a concentration of about 0.5 to 20%
w/w, for example about 0.5 to 10% w/w, for example about 1.5%
w/w.
[0189] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0190] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising for example cocoa
butter or a salicylate.
[0191] Formulations suitable for intrapulmonary or nasal
administration have a particle size for example in the range of 0.1
to 500 microns (including particle sizes in a range between 0.1 and
500 microns in increments microns such as 0.5, 1, 30 microns, 35
microns, etc.), which is administered by rapid inhalation through
the nasal passage or by inhalation through the mouth so as to reach
the alveolar sacs. Suitable formulations include aqueous or oily
solutions of the active ingredient. Formulations suitable for
aerosol or dry powder administration may be prepared according to
conventional methods and may be delivered with other therapeutic
agents such as compounds heretofore used in the treatment or
prophylaxis disorders as described below.
[0192] The formulations may be packaged in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water, for
injection immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Preferred
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of the active ingredient.
[0193] The subject matter further provides veterinary compositions
comprising at least one active ingredient as above defined together
with a veterinary carrier therefore. Veterinary carriers are
materials useful for the purpose of administering the composition
and may be solid, liquid or gaseous materials which are otherwise
inert or acceptable in the veterinary art and are compatible with
the active ingredient. These veterinary compositions may be
administered parenterally, orally or by any other desired
route.
[0194] In particular embodiments the pharmaceutical composition
comprising the presently disclosed compounds further comprise a
chemotherapeutic agent.
IV. Methods
[0195] The methods described herein provide methods for selectively
inhibiting bacterial .beta.-glucuronidases. In the methods, an
effective amount of at least one selective .beta.-glucuronidase
inhibitor can be administered to a subject in need thereof. That
is, a subject being treated with a chemotherapeutic agent, such as
a camptothecin-derived antineoplastic agent or
glucuronidase-substrate agents or compounds.
[0196] Also described herein are methods for improving efficacy of
camptothecin-derived antineoplastic agents or
glucuronidase-substrate agents or compounds by attenuating
reactivation by bacterial .beta.-glucuronidases of glucuronidated
metabolites of camptothecin-derived antineoplastic agents or
glucuronidase-substrate agents or compounds. In the methods, a
therapeutically effective amount of at least one selective
.beta.-glucuronidase inhibitor can be administered to a subject
having or about to have treatment with a chemotherapeutic agent,
particularly a camptothecin-derived antineoplastic agent or any
other glucuronidase-substrate agents or compounds.
[0197] The compounds disclosed herein can be used in compositions
and methods for inhibiting bacterial .beta.-glucuronidases and for
improving efficacy of camptothecin-derived antineoplastic agents or
glucuronidase-substrate agents or compounds by attenuating the
gastrointestinal distress caused by reactivation of glucuronidated
metabolites of such agents. The presently disclosed compounds find
use in selectively inhibiting the activity of one or more microbial
beta-glucuronidase (GUS) enzymes found in the mammalian GI tract.
The presently disclosed compounds reduce, inhibit, or otherwise
diminish the activity of one or more GUS enzymes. The presently
disclosed compounds may or may not be a specific GUS inhibitor.
However, the GUS inhibitor is selective for microbial or bacterial
beta-glucuronidase as compared to other non-microbial
beta-glucuronidases. The methods comprise contacting GUS with an
effective amount of a compound of Formula I. Such methods include
the following.
[0198] The compounds and compositions can be used in methods for
treating cancer to reduce the side effects of antineoplastic
agents, such as camptothecin-derived antineoplastic agents. Thus,
the gastrointestinal distress that typically accompanies treatment
with an antineoplastic agent can be attenuated. The methods are
also useful for attenuating or improving any adverse reactions
associated with administration of glucuronidase-substrate agent(s)
or compound(s). Methods of the present invention include
administering to a subject in need thereof a therapeutically
effective amount of at least one .beta.-glucuronidase inhibiting
agent that selectively inhibits bacterial .beta.-glucuronidases
from hydrolyzing glucuronides.
[0199] A method of ameliorating a side effect in a human in need
thereof, comprising administering to said human an effective amount
of a compound of Formula I.
[0200] A method of inhibiting a GUS comprising, contacting a GUS
with a compound of Formula I.
[0201] By "contact" is intended bringing the compound within close
enough proximity to the target GUS such that the compound is able
to inhibit the activity of GUS. The compound can be contacted with
GUS in vitro or in vivo via administration of the compound to a
subject.
[0202] In embodiments, the methods are directed to:
[0203] A method for selectively inhibiting bacterial
.beta.-glucuronidases, the method comprising administering to a
subject in need thereof an effective amount of at least one
compound of Formula I. In embodiments, the bacteria are selected
from the group consisting of a Bacteroides sp., Bifidobacterium
sp., Catenabacterium sp., Clostridium sp., Corynebacterium sp.,
Enterococcus faecalis, Enterobacteriaceae, Lactobacillus sp.,
Peptostreptococcus sp., Propionibacterium sp., Proteus sp.,
Mycobacterium sp., Pseudomonas sp., Staphylococcus sp. and
Streptococcus sp.
[0204] A method for improving camptothecin-derived antineoplastic
agent efficiency, the method comprising administering to a subject
prior to, concurrently with or after administration of a
camptothecin-derived antineoplastic agent a therapeutically
effective amount of at least one compound of Formula I.
[0205] A method for attenuating side effects in a subject being
administered a camptothecin-derived antineoplastic agent, the
method comprising administering prior to, concurrently with or
after administration of a camptothecin-derived antineoplastic agent
a therapeutically effective amount of at least one compound of
Formula I.
[0206] In embodiments, the camptothecin-derived antineoplastic
agent is selected from the group consisting of camptothecin,
diflomotecan, exatecan, gimatecan, irinotecan, karenitecin,
lurtotecan, rubitecan, silatecan and topotecan. In particular, the
camptothecin-derived antineoplastic agent is irinotecan.
[0207] A method to alleviate gastrointestinal distress associated
with chemotherapy comprising: a) administering to an animal an
anti-cancer effective amount of a chemotherapeutic agent, and b)
administering to the same animal an inhibitory effective amount of
a one compound of Formula I. In an aspect of this embodiment, the
chemotherapeutic active agent is a camptothecin-derived
antineoplastic agent.
[0208] A method for improving the efficiency of a
glucuronidase-substrate agent or compound, the method comprising
administering to a subject prior to, concurrently with or after
administration of said agent or compound a therapeutically
effective amount of at least one compound of Formula I.
[0209] Methods for assessing .beta.-glucuronidase activity are
known in the art. See, e.g., Farnleitner et al. (2002) Water Res.
36:975-981; Fior et al. (2009) Plant Sci. 176:130-135; and Szasz
(1967) Clin. Chem. 13:752-759. One of skill in the art is familiar
with assays for testing the ability of an active compound for
selectively inhibiting .beta.-glucuronidases with minor or no
toxicity to the bacteria that inhabit the gastrointestinal tract.
.beta.-glucuronidase activity of bacteria provided the selective
.beta.-glucuronidase inhibitor can be reduced by at least about 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% when compared
to bacteria not provided the selective .beta.-glucuronidase
inhibitor.
[0210] In one aspect, provided herein is a method for ameliorating
the side effects, in particular the GI side effects, in the
treatment of cancer in a subject in need thereof. The term "cancer"
refers to or describe the physiological condition in mammals that
is typically characterized by unregulated cell growth. Examples of
cancer include, but are not limited to, carcinoma, lymphoma,
blastoma, sarcoma, and leukemia or lymphoid malignancies. More
particular examples of such cancers include melanoma, squamous cell
cancer (e.g., epithelial squamous cell cancer), lung cancer
including melanoma, multiple myeloma, small-cell lung cancer,
non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung
and squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma,
glioblastoma multiforme, KRAS mutant solid tumors, indolent
non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), diffuse
large B-cell lymphoma, thyroid cancer, non-Hodgkin's lymphoma,
basal cell carcinoma, hematological tumors, B-cell non-Hodgkin's
lymphoma, acute myeloid leukemia (AML), cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
including triple negative breast cancer, colon cancer, rectal
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma, as well as head and neck cancer. Also included
are "hematological malignancies" or "hematological cancer," which
are the types of cancer that affect blood, bone marrow, and lymph
nodes. Hematological malignancies may derive from either of the two
major blood cell lineages: myeloid and lymphoid cell lines. The
myeloid cell line normally produces granulocytes, erythrocytes,
thrombocytes, macrophages and mast cells; the lymphoid cell line
produces B, T, NK and plasma cells. Lymphomas, lymphocytic
leukemias, and myeloma are from the lymphoid line, while acute and
chronic myelogenous leukemia, myelodysplastic syndromes and
myeloproliferative diseases are myeloid in origin. Leukemias
include Acute lymphoblastic leukemia (ALL), Acute myelogenous
leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronic
myelogenous leukemia (CML), Acute monocytic leukemia (AMOL) and
small lymphocytic lymphoma (SLL). Lymphomas include Hodgkin's
lymphomas (all four subtypes) and Non-Hodgkin's lymphomas (NHL, all
subtypes).
[0211] The presently disclosed compounds may be administered in any
suitable manner known in the art. In some embodiments, the compound
of Formula I, or a pharmaceutically acceptable salt thereof, is
administered intravenously, intramuscularly, subcutaneously,
topically, orally, transdermally, intraperitoneally,
intraorbitally, by implantation, by inhalation, intrathecally,
intraventricularly, intratumorally, or intranasally.
[0212] In some embodiments, the compound of Formula I is
administered continuously. In other embodiments, the compound of
Formula I is administered intermittently. Moreover, treatment of a
subject with an effective amount of a compound of Formula I can
include a single treatment or can include a series of
treatments.
[0213] It is understood that appropriate doses of the active
compound depends upon a number of factors within the knowledge of
the ordinarily skilled physician or veterinarian. The dose(s) of
the active compound will vary, for example, depending upon the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, and any drug combination.
[0214] It will also be appreciated that the effective dosage of a
compound of Formula I or a pharmaceutically acceptable salt used
for treatment may increase or decrease over the course of a
particular treatment. Changes in dosage may result and become
apparent from the results of diagnostic assays.
[0215] In some embodiments, a compound of Formula I is administered
to the subject at a dose of between about 0.001 .mu.g/kg and about
1000 mg/kg, including but not limited to about 0.001 .mu.g/kg, 0.01
.mu.g/kg, 0.05 .mu.g/kg, 0.1 .mu.g/kg, 0.5 .mu.g/kg, 1 .mu.g/kg, 10
.mu.g/kg, 25 .mu.g/kg, 50 .mu.g/kg, 100 .mu.g/kg, 250 .mu.g/kg, 500
.mu.g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 100
mg/kg, and 200 mg/kg.
[0216] In some embodiments, the subject that is administered a
compound of Formula I is a mammal, such as domesticated animals
(e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans
and non-human primates such as monkeys), rabbits, and rodents
(e.g., mice and rats). In some embodiments, the subject treated is
a human.
[0217] In some embodiments, the methods for ameliorating the side
effects or improving the efficacy of a chemotherapeutic drug
comprising administering to the subject an effective amount of a
compound of Formula I, or a pharmaceutically acceptable salt
thereof, further comprises administering the chemotherapeutic drug.
For example, a compound of Formula I and a chemotherapeutic agent
may be administered sequentially (at different times) or
concurrently (at the same time). A compound of Formula I and
chemotherapeutic agent may be administered by the same route of
administration or by different routes of administration.
[0218] The chemotherapeutic agent can be a camptothecin-derived
antineoplastic agent. As used herein, "a camptothecin-derived
antineoplastic agent" and the like means a cytotoxic quinoline
alkaloid that inhibits the DNA enzyme topoisomerase I.
Camptothecin-derived antineoplastic agents include, but are not
limited to, camptothecin (i.e.,
(S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoli-
ne-3,14-(4H,12H)-dione); diflomotecan (i.e.,
(5R)-5-ethyl-9,10-difluoro-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3-
',4':6,7]indolizino[1,2-b]quinoline-3,15-dione); exatecan (i.e.,
(1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-meth-
yl-10H,
13H-benzo(de)pyrano(3',4':6,7)indolizino(1,2-b)quinoline-10,
13-dione); gimatecan (i.e.,
(4S)-11-((E)-((1,1-dimethylethoxy)imino)methyl)-4-ethyl-4-hydroxy-1,12-di-
hydro-14H-pyrano(3',4':6,7)indolizino(1,2-b)quinoline-3,14(4H)-dion
e); irinotecan (i.e.,
(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',4'-
:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'bipiperidine]-1'-carboxylate);
karenitecin (i.e.,
(4S)-4-ethyl-4-hydroxy-11-(2-trimethylsilyl)ethyl)-1H-pyrano[3',4':6,7]in-
dolizino[1,2-b]quinoline-3,14(4H,12H)-dione); lurtotecan (i.e.,
7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin);
rubitecan (i.e.,
(4S)-4-ethyl-4-hydroxy-10-nitro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quin-
oline-3,14(4H, 12H)-dione); silatecan (i.e.,
7-tert-butyldimethylsilyl-10-hydroxycamptothecin); and topotecan
(i.e.,
(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]-
indolizino[1,2-b]quinoline-3,14(4H, 12H)-dione).
[0219] Of particular interest is irinotecan (CPT-11 or
Camptosan.RTM.), which is a potent camptothecin-derived
antineoplastic agent for treating solid malignancies of the brain,
colon and lung, as well as refractory forms of leukemia and
lymphoma. Irinotecan is a prodrug that must be converted into its
active form, SN-38 (7-ethyl-10-hydroxy-camptothecin), to have
antineoplastic activity. During its excretion, SN-38 is
glucuronidated to SN-38 glucuronide (SN-38G) by phase II drug
metabolizing UDP-glucurono syltransferases.
[0220] The term "glucuronidase-substrate agent(s) or compound(s)"
refers to any drug, agent or compound or, in particular, a
metabolite thereof that can be a substrate for glucuronidase. Thus,
in some instances, a drug, compound or agent that is not itself a
substrate, but is metabolized to a substrate is encompassed by the
term as used herein. Any drug, compound or agent or metabolite
thereof that is glucuronidated, also referred to as glucuronides,
can be a substrate for glucuronidase and is also described herein
as glucuronidase-substrate agent(s) or compound(s). Many drugs,
agents or compounds undergo glucuronidation at some point in their
metabolism. Alternatively, the drug, agent or compound may be a
glucuronide pro-drug. These glucuronides may have different
properties than the parent drug, agent or compound. Glucuronidation
can modulate the potency of some drugs: the 6-glucuronide of
morphine is a more potent analgesic than the parent compound,
whereas the 3-glucuronide is a morphine antagonist. In addition,
steroid glucuronidation can produce more active or toxic
metabolites under pathophysiological conditions or during steroid
therapies.
[0221] Drugs, agents or compounds or metabolites thereof which are
substrates for glucuronidase can have their respective properties
altered by glucuronidase hydrolysis. In a specific, non-limiting
example, if the drug, agent, compound or metabolite thereof has
been metabolized to a glucuronide, the hydrolysis of the
glucuronide can reactivate the drug, agent, compound or metabolite
thereof. In many cases, this reactivation can cause adverse
reactions. For example, if a glucuronide drug, agent or compound or
metabolite thereof is present in the gut, glucuronidase hydrolysis
in the gut can lead to gastrointestinal distress.
[0222] The methods described herein are useful for attenuating,
ameliorating or improving the adverse reactions, such as
gastrointestinal distress, caused by the action of glucuronidase on
a drug, agent or compound or, in particular, a metabolite thereof.
As described fully elsewhere herein, hydrolysis of glucuronides can
lead to adverse reactions. The methods described herein inhibit or
decrease the activity of .beta.-glucuronidases. The methods can
therefore be useful to attenuate, ameliorate or improve adverse
reactions, such as gastrointestinal distress, associated with
administering such drugs, agents or compounds. The methods can also
improve the tolerance of any such drug, agent or compound or
metabolite thereof that can form a glucuronide. As such,
administration of a glucuronidase inhibitor can rescue or improve a
treatment with any drug, agent or compound, wherein glucuronidase
hydrolysis of a glucuronide related to the drug, agent, compound or
metabolite thereof is causing one or more adverse reactions,
particularly gastrointestinal distress or toxicity. Patient
compliance and outlook would also improve with the lessening of
adverse reactions.
[0223] As mentioned above, the term "glucuronidase-substrate
agent(s) or compound(s)" refers to any drug, agent or compound or,
in particular, a metabolite thereof that can be a substrate for
glucuronidase. These can include any chemical compound useful in
the treatment of disease, for example, but not limited to, cancer.
Examples of such chemotherapeutic agents include NSAIDs, sorafenib,
alkylating agents such as thiotepa and cyclophosphamide
(CYTOXAN.RTM.); alkyl sulfonates such as busulfan, improsulfan, and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; pemetrexed; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
podophyllotoxin; podophyllinic acid; teniposide; cryptophycins
(particularly cryptophycin 1 and cryptophycin 8); dolastatin;
duocarmycin (including the synthetic analogues, KW-2189 and
CB1-.TM.1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral
alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen
mustards such as chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;
antibiotics such as the enediyne antibiotics (e. g., calicheamicin,
especially calicheamicin gammalI and calicheamicin omegaIl (see,
e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186
(1994)); dynemicin, including dynemicin A; an esperamicin; as well
as neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores), aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, carabicin, carminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including
ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.) and deoxydoxorubicin),
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such
as mitomycin C, mycophenolic acid, nogalamycin, olivomycins,
peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin,
zorubicin; anti-metabolites such as methotrexate, gemcitabine
(GEMZAR.RTM.), tegafur (UFTORAL.RTM.), capecitabine (XELODA.RTM.),
an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, and floxuridine; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex
(JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL.RTM.),
albumin-engineered nanoparticle formulation of paclitaxel
(ABRAXANE.TM.), and doxetaxel (TAXOTERE.RTM.); chloranbucil;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine (VELBAN.RTM.); platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine
(ONCOVIN.RTM.); oxaliplatin; leucovovin; vinorelbine
(NAVELBINE.RTM.); novantrone; edatrexate; daunomycin; aminopterin;
ibandronate; topoisomerase inhibitor RFS 2000;
difluorometlhylornithine (DMFO); retinoids such as retinoic acid;
pharmaceutically acceptable salts, acids or derivatives of any of
the above; as well as combinations of two or more of the above such
as CHOP, an abbreviation for a combined therapy of
cyclophosphamide, doxorubicin, vincristine, and prednisolone, and
FOLFOX, an abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0224] Additional examples of chemotherapeutic agents include
anti-hormonal agents that act to regulate, reduce, block, or
inhibit the effects of hormones that can promote the growth of
cancer, and are often in the form of systemic, or whole-body
treatment. They may be hormones themselves. Examples include
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene (EVISTA.RTM.), droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene (FARESTON.RTM.); anti-progesterones; estrogen
receptor down-regulators (ERDs); estrogen receptor antagonists such
as fulvestrant (FASLODEX.RTM.); agents that function to suppress or
shut down the ovaries, for example, leutinizing hormone-releasing
hormone (LHRH) agonists such as leuprolide acetate (LUPRON.RTM. and
ELIGARD.RTM.), goserelin acetate, buserelin acetate and
tripterelin; anti-androgens such as flutamide, nilutamide and
bicalutamide; and aromatase inhibitors that inhibit the enzyme
aromatase, which regulates estrogen production in the adrenal
glands, such as, for example, 4(5)-imidazoles, aminoglutethimide,
megestrol acetate (MEGASE.RTM.), exemestane (AROMASIN.RTM.),
formestanie, fadrozole, vorozole (RIVISOR.RTM.), letrozole
(FEMARA.RTM.), and anastrozole (ARIMIDEX.RTM.). In addition, such
definition of chemotherapeutic agents includes bisphosphonates such
as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.), etidronate
(DIDROCAL.RTM.), NE-58095, zoledronic acid/zoledronate
(ZOMETA.RTM.), alendronate (FOSAMAX.RTM.), pamidronate
(AREDIA.RTM.), tiludronate (SKELID.RTM.), or risedronate
(ACTONEL.RTM.); as well as troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); anti-sense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in abherant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); an anti-estrogen such as
fulvestrant; EGFR inhibitor such as erlotinib or cetuximab; an
anti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH (e.g.,
ABARELIX.RTM.); 17AAG (geldanamycin derivative that is a heat shock
protein (Hsp) 90 poison), and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0225] Also included in the definition of "chemotherapeutic agent"
are: (i) anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (including NOLVADEX.RTM.; tamoxifen citrate), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. (toremifine citrate); (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. (megestrol
acetate), AROMASIN.RTM. (exemestane; Pfizer), formestanie,
fadrozole, RIVISOR.RTM. (vorozole), FEMARA.RTM. (letrozole;
Novartis), and ARIMIDEX.RTM. (anastrozole; AstraZeneca); (iii)
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase
inhibitors; (v) lipid kinase inhibitors; (vi) antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in aberrant cell
proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;
(vii) ribozymes such as VEGF expression inhibitors (e.g.,
ANGIOZYME.RTM.) and HER2 expression inhibitors; (viii) vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.,
LEUVECTIN.RTM., and VAXID.RTM.; PROLEUKIN.RTM. rIL-2; a
topoisomerase 1 inhibitor such as LURTOTECAN.RTM.; ABARELIX.RTM.
rmRH; (ix) anti-angiogenic agents such as bevacizumab
(AVASTIN.RTM., Genentech); and (x) pharmaceutically acceptable
salts, acids and derivatives of any of the above.
V. Preparation of Formula I Compounds
[0226] Compounds of Formula I may be synthesized by synthetic
routes that include processes analogous to those well-known in the
chemical arts, particularly in light of the description contained
herein, and those for other heterocycles described in:
Comprehensive Heterocyclic Chemistry II, Editors Katritzky and
Rees, Elsevier, 1997, e.g. Volume 3; Liebigs Annalen der Chemie,
(9):1910-16, (1985); Helvetica Chimica Acta, 41:1052-60, (1958);
Arzneimittel-Forschung, 40(12):1328-31, (1990), each of which are
expressly incorporated by reference. Starting materials are
generally available from commercial sources such as Aldrich
Chemicals (Milwaukee, Wis.) or are readily prepared using methods
well known to those skilled in the art (e.g., prepared by methods
generally described in Louis F. Fieser and Mary Fieser, Reagents
for Organic Synthesis, v. 1-23, Wiley, N.Y. (1967-2006 ed.), or
Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed.
Springer-Verlag, Berlin, including supplements (also available via
the Beilstein online database).
[0227] Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
Formula I compounds and necessary reagents and intermediates are
known in the art and include, for example, those described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 3.sup.rd Ed., John Wiley and Sons (1999); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995) and subsequent editions thereof.
[0228] Compounds of Formula I may be prepared singly or as compound
libraries comprising at least 2, for example 5 to 1,000 compounds,
or 10 to 100 compounds. Libraries of compounds of Formula I may be
prepared by a combinatorial `split and mix` approach or by multiple
parallel syntheses using either solution phase or solid phase
chemistry, by procedures known to those skilled in the art. Thus,
according to a further aspect, there is provided a compound library
comprising at least 2 compounds, or pharmaceutically acceptable
salts thereof.
[0229] The General Procedures and Examples provide exemplary
methods for preparing Formula I compounds. Those skilled in the art
will appreciate that other synthetic routes may be used to
synthesize the Formula I compounds. Although specific starting
materials and reagents are depicted and discussed in the Schemes,
General Procedures, and Examples, other starting materials and
reagents can be easily substituted to provide a variety of
derivatives and/or reaction conditions. In addition, many of the
exemplary compounds prepared by the described methods can be
further modified in light of this disclosure using conventional
chemistry well known to those skilled in the art.
[0230] In preparing compounds of Formulas I, protection of remote
functionality (e.g., primary or secondary amine) of intermediates
may be necessary. The need for such protection will vary depending
on the nature of the remote functionality and the conditions of the
preparation methods. Suitable amino-protecting groups include
acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl
(CBz or CBZ) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). The need
for such protection is readily determined by one skilled in the
art. For a general description of protecting groups and their use,
see T. W. Greene, Protective Groups in Organic Synthesis, John
Wiley & Sons, New York, 1991.
[0231] The following examples are offered by way of illustration
and not by way of limitation.
Examples
Example 1. Synthesis of Inh9 and Compounds 2-8
##STR00027##
[0232] Compounds 2-8 in Table 1 were prepared by a similar method
as Inh9 from intermediate 4.
Synthesis of Intermediate 1
[0233] To a solution of cyclohexanone (5.0 g, 5.3 mL, 51 mmol, 1.0
eq.) in 16 mL of methanol was slowly added carbon disulfide (7.8 g,
6.1 mL, 102 mmol, 2.0 eq.) and malononitrile (3.4 g, 51 mmol, 1.0
eq.), and the mixture was stirred for 5 minutes while maintaining
the temperature below 20.degree. C. Triethylamine (2.5 mL) was
added, and the reaction mixture was stirred overnight at rt.
Precipitated product was filtered, washed with methanol and vacuum
dried to provide intermediate 1 as an orange solid (4.3 g,
40%).
[0234] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.69 (s, 2H),
2.68 (m, 2H), 2.59 (m, 2H), 1.68 (m, 4H)
[0235] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.9 min. MS (ESI+): 223
[M+H].sup.+
Synthesis of Intermediate 2
[0236] To a suspension of intermediate 1 (4.0 g, 18 mmol, 1.0 eq.)
in ethanol (15 mL) was added morpholine (8 mL, 90 mmol, 5 eq.) and
the mixture was heated under reflux overnight. Precipitated product
was cooled to rt, degassed with nitrogen, filtered, washed with
ethanol and vacuum dried to obtain intermediate 2 as an orange
solid (3.8 g, 76%).
[0237] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.33-4.27 (m,
1H), 3.74 (m, 4H), 3.67 (m, 4H), 3.55-3.50 (m, 1H), 2.61 (t, J=5.8
Hz, 2H), 2.41 (t, J=5.8 Hz, 2H), 1.77-1.65 (m, 2H), 1.62-1.52 (m,
2H)
[0238] LC-MS (.lamda.=254 nm): 99%, t.sub.R=4.9 min. MS (ESI+): 276
[M+H].sup.+
Synthesis of Intermediate 3
[0239] To a solution of intermediate 2 (3.0 g, 11 mmol, 1.0 eq.) in
22 mL of DMF was added 2-chloroacetonitrile (0.8 mL, 11.99 mmol,
1.1 eq.) and stirred at rt for an hour. Then the first portion of
aq. KOH (10% w/v, 5.5 mL) was added to the reaction mixture and it
was stirred at rt overnight, after which a second portion of aq.
KOH (10% w/v, 5.5 mL) was added to the reaction mixture and stirred
for another 4 hours at rt. Water (50 mL) was added to the
precipitated solid product after which it was filtered and vacuum
dried to obtain intermediate 3 as a pinkish yellow solid (2.36 g,
69%).
[0240] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.35 (s, 2H),
3.84-3.70 (m, 4H), 3.27 (t, J=6.5 Hz, 2H), 3.19-3.10 (m, 4H), 2.64
(t, J=5.7 Hz, 2H), 1.88-1.75 (m, 2H), 1.70-1.58 (m, 2H)
[0241] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.9 min. MS (ESI+): 315
[M+H].sup.+
Synthesis of Intermediate 4
[0242] A solution of sodium nitrite (1.5 g, 22 mmol, 3.0 eq.) in
water (7.3 mL) was added dropwise, over 30 minutes to a suspension
of intermediate 3 (2.3 g, 7.3 mmol, 1.0 eq.) in conc. HCl acid (15
mL) at 0-5.degree. C. The mixture was stirred for an hour at
0-5.degree. C. and then allowed to stir at rt overnight. Water (100
mL) was added to the precipitated product after which it was
filtered, washed with water, and vacuum dried to obtain
intermediate 4 as a yellow solid (2.4 g, 92%).
[0243] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 3.93-3.85 (m,
2H), 3.75 (t, J=6.6 Hz, 1H), 3.48-3.41 (m, 2H), 2.74 (t, J=5.9 Hz,
1H), 2.08-1.97 (m, 1H), 1.88-1.77 (m, 1H)
[0244] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.4 min. MS (ESI+): 362
[M+H].sup.+
Synthesis of Intermediate 5
[0245] To a solution of intermediate 4 (85 mg, 0.23 mmol, 1.0 eq.)
in CH.sub.3CN (2 mL) was added K.sub.2CO.sub.3 (300 mg) and
1-Boc-piperazine (110 mg, 0.59 mmol, 2.5 eq.) and the reaction
mixture was heated under reflux overnight. Upon completion, the
reaction was quenched by addition of 20 mL of sat. NaHCO.sub.3
solution. The aqueous phase was extracted with CH.sub.2Cl.sub.2
(3.times.20 mL) and the organic layers were combined, washed with
brine, dried over anhydrous Na.sub.2SO.sub.4, and filtered. Solvent
was removed by rotary evaporation to obtain a dark brown crude
material. The crude material was adsorbed onto silica gel and
purified by normal phase automated Teledyne Isco chromatography
using a CH.sub.2C.sub.12/MeOH/NH.sub.3 solvent system. Intermediate
5 was obtained as a pale yellow solid (50 mg, 43%).
[0246] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 4.08-4.02 (m,
4H), 3.92-3.83 (m, 4H), 3.77 (t, J=6.7 Hz, 2H), 3.68-3.60 (m, 4H),
3.38-3.30 (m, 4H), 2.73 (t, J=5.8 Hz, 2H), 2.03-1.94 (m, 2H),
1.85-1.76 (m, 2H), 1.49 (s, 9H)
[0247] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.5 min. MS (ESI+): 512
[M+H].sup.+
Synthesis of Inh9
[0248] To a solution of intermediate 5 (50 mg, 0.098 mmol) in
CH.sub.2Cl.sub.2 (1 mL) at 0.degree. C. was added trifluoroacetic
acid (1 mL) and the reaction mixture was stirred until completion
at room temperature. The solution was washed with saturated
NaHCO.sub.3 solution, dried over anhydrous Na.sub.2SO.sub.4,
filtered, and vacuum concentrated to obtain Inh9 as a yellow solid
(40 mg, 99%).
[0249] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 9.91 (s, 1H),
4.36 (s, 4H), 3.92-3.79 (m, 4H), 3.68 (t, J=6.2 Hz, 2H), 3.48 (s,
3H), 3.40-3.31 (m, 4H), 2.71 (t, J=5.6 Hz, 2H), 1.97 (m, 2H), 1.79
(m, 2H)
[0250] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.0 min. MS (ESI+): 412
[M+H].sup.+
Example 2. Synthesis of Compounds 12-17
##STR00028##
[0252] Intermediates 6 and 7 were synthesized according to the
literature (J. C. A. Hunt et al. Bioorg. Med. Chem. Lett. 17 (2007)
5222-5226).
Compounds 12-14 and 16-17 in Table 1 were prepared by a similar
method as compound 15 from intermediate 7.
Synthesis of Intermediate 8
[0253] Intermediate 7 (500 mg, 2.04 mmol, 1.0 eq.) was added
portion-wise to a stirred suspension of sodium hydride (60%
dispersion in mineral oil, 85 mg, 2.12 mmol, 1.1 eq.) in DMF (10.3
mL) under nitrogen at 0.degree. C. The reaction mixture was stirred
at room temperature for 35-40 min. To the resultant solution was
added N-phenyl-trifluoromethanesulfonimide (729 mg, 2.04 mmol, 1.0
eq.) in one portion. After 20 min., dimethylamine (1.06 mL, 2.12
mmol, 1.1 eq.) was added to the reaction mixture and stirred
overnight. The reaction was slowly quenched with water (5 mL) and
extracted with ethyl acetate (3.times.20 mL). The combined organic
fractions were washed with saturated NaHCO.sub.3 solution, dried
over anhydrous Na.sub.2SO.sub.4, filtered, concentrated under
reduced pressure, and purified by reverse phase automated Teledyne
Isco chromatography using a MeOH/H.sub.2O/0.1% acetic acid solvent
system. Intermediate 8 was obtained as pale yellow solid (255 mg,
46%).
[0254] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.22 (t, J=6.5
Hz, 2H), 2.85 (s, 6H), 2.59 (t, J=5.8 Hz, 2H), 1.84-1.72 (m, 2H),
1.60 (m, 2H)
[0255] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.1 min. MS (ESI+): 273
[M+H].sup.+
Synthesis of Intermediate 9
[0256] A solution of sodium nitrite (266 mg, 3.9 mmol, 3.0 eq.) in
water (2 mL) was added dropwise over 30 minutes to a suspension of
intermediate 8 (350 mg, 1.3 mmol, 1.0 eq.) in conc. HCl acid (10
mL) at 0-5.degree. C. The mixture was stirred for an hour at
0-5.degree. C. and then allowed to stir at rt overnight. Water (100
mL) was added to the precipitated product after which it was
filtered, washed with water, and vacuum dried to obtain
intermediate 9 as a pale yellow solid (291 g, 71%).
[0257] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 3.68 (t, J=6.6
Hz, 2H), 3.09 (s, 4H), 2.72 (t, J=5.9 Hz, 2H), 2.04-1.93 (m, 2H),
1.81-1.72 (m, 2H)
[0258] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.8 min. MS (ESI+): 320
[M+H].sup.+
Synthesis of Intermediate 10
[0259] To a solution of intermediate 4 (150 mg, 0.47 mmol, 1.0 eq.)
in CH.sub.3CN (4 mL) was added K.sub.2CO.sub.3 (300 mg) and
1-Boc-piperazine (219 mg, 1.17 mmol, 2.5 eq.) and the reaction
mixture was heated under reflux overnight. Upon completion, the
reaction was quenched by addition of 20 mL of sat. NaHCO.sub.3
solution. The aqueous phase was extracted with CH.sub.2Cl.sub.2
(3.times.20 mL) and the organic layers were combined, washed with
brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to obtain a dark brown crude
material. The crude material was adsorbed onto silica gel, and
purified by normal phase automated Teledyne Isco chromatography
using a CH.sub.2C.sub.12/MeOH/NH.sub.3 solvent system. Intermediate
10 was obtained as a white solid (50 mg, 23%).
[0260] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 4.07-3.98 (m,
4H), 3.72 (t, J=6.6 Hz, 2H), 3.62 (dd, J=6.1, 4.3 Hz, 4H), 3.01 (s,
6H), 2.71 (t, J=5.9 Hz, 2H), 2.01-1.89 (m, 2H), 1.76 (m, 2H), 1.48
(s, 9H)
[0261] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.9 min. MS (ESI+): 470
[M+H].sup.+
Synthesis of Compound 15
[0262] To a solution of intermediate 10 (50 mg, 0.106 mmol) in
CH.sub.2Cl.sub.2 (1 mL) at 0.degree. C. was added trifluoroacetic
acid (1 mL), and the reaction mixture was stirred until completion
at room temperature. The solution was washed with saturated
NaHCO.sub.3 solution, dried over anhydrous Na.sub.2SO.sub.4,
filtered, and vacuum concentrated to obtain compound 15 as a white
solid (35 mg, 90%).
[0263] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.15-4.06 (m,
4H), 3.59 (t, J=6.5 Hz, 2H), 3.29-3.21 (m, 4H), 3.00 (s, 6H), 2.73
(t, J=5.8 Hz, 2H), 1.98-1.87 (m, 2H), 1.77-1.64 (m, 2H)
[0264] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.6 min. MS (ESI+): 370
[M+H].sup.+
Example 3. Synthesis of Compound 18
##STR00029##
[0265] Example 4. Synthesis of Compounds 21, 28 and 29
##STR00030##
[0266] Compounds 28 and 29 in Table were prepared by a similar
method as compound 21.
Synthesis of Intermediate 11
[0267] 2,6-Dichloropyrdiatne-3-carbonitrile (300 g, 1.73 mmol, 1.0
eq.) was dissolved in DMF (2 mL) in a flame dried round bottom
flask under nitrogen atmosphere. To the resultant solution was
added morpholine (0.15 me, 1.73 mmol, 1.0 eq.), and triethylamine
(0.5 mL, 3.46 mmol, 2.0 eq.). The reaction was stirred overnight at
room temperature under nitrogen. The reaction was quenched with
satur aterqueous NH.sub.4C solution (10 mL). The aqueous phase was
separated and extracted with EtOAc (5.times.10 mL). All combined
organic layers were dried over anhydrous Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure to obtain a white
crude material. .sup.1H NMR and LC-MS of the crude material showed
the presence both mono- and di-amination products (6:1, same
t.sub.R). The crude material was used in the next reaction without
further purification (350 mg).
Synthesis of Intermediate 12
[0268] Crude intermediate 11 (350 mg) was dissolved in ethanol (9
mL) in a flame dried round bottom flask under an atmosphere of
nitrogen. To the resultant solution was added 2-mercaptoacetamide
(100 mg/l mL in methanolic ammonia solution, 1.71 mL, 1.88 mmol)
and anhydrous K.sub.2CO.sub.3 (521 mg, 3.8 mmol). The reaction was
refluxed overnight at 100.degree. C. under nitrogen. The reaction
was quenched with water (50 mL) which precipitated the desired
product. The precipitated product was filtered, washed with water
(50 mL), and concentrated under reduced pressure to obtain
intermediate 12 as a pale yellow solid (299 mg, 68% over two
steps).
[0269] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.11 (d, J=9.1
Hz, 1H), 7.03 (brs, 2H), 6.93 (d, J=9.1 Hz, 1H), 6.82 (brs, 2H),
3.73-3.67 (m, 4H), 3.59-3.53 (m, 4H)
[0270] LC-MS (.lamda.=254 nm): 99%, t.sub.R=4.6 min. MS (ESI+): 279
[M+H].sup.+
Synthesis of Intermediate 13
[0271] Intermediate 12 (150 mg, 1.56 mmol, 1.0 eq.) was dissolved
in dioxane (2 mL) in a flame dried round bottom flask under
nitrogen atmosphere. To the resultant solution was added
trichloromethyl chloroformate (0.07 mL, 0.61 mmol, 1.1 eq.), and
the reaction was refluxed for 3 hours at 102.degree. C. under
nitrogen. The reaction was slowly quenched with water (10 mL). The
precipitated product was filtered, washed with water (50 mL), and
concentrated under reduced pressure to obtain a yellow solid (118
mg, 69%).
[0272] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 12.03 (brs, 1H),
11.29 (brs, 1H), 8.33 (d, J=9.3 Hz, 1H), 7.10 (d, J=9.3 Hz, 1H),
3.70 (m, 4H), 3.68-3.60 (m, 4H)
[0273] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.0 min. MS (ESI+): 305
[M+H].sup.+
[0274] The above yellow solid (110 mg, 0.36 mmol, 1.0 eq.) was
added to phenylphosphonic dichloride (4 mL) in a flame dried round
bottom flask under nitrogen atmosphere. The reaction was refluxed
for 2 hours at 170.degree. C. under nitrogen. The reaction was
slowly quenched with water (10 mL) which precipitated the desired
product. The precipitated product was filtered, washed with more
water (30 mL), and concentrated under reduced pressure to obtain
intermediate 13 as a yellow solid (>30 mg, >25%).
[0275] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.34 (d, J=9.1
Hz, 1H), 6.80 (d, J=9.1 Hz, 1H), 3.85-3.75 (m, 8H)
[0276] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.6 min. MS (ESI+): 342
[M+H].sup.+
Synthesis of Intermediate 14
[0277] To a solution of intermediate 13 (90 mg, 0.088 mmol, 1.0
eq.) in CH.sub.3CN (2 mL) was added K.sub.2CO.sub.3 (300 mg) and
1-Boc-piperazine (82 mg, 0.43 mmol, 5.0 eq.), and the reaction
mixture was heated under reflux overnight. Upon completion, the
reaction was quenched by addition of 20 mL of sat. NaHCO.sub.3
solution. The aqueous phase was extracted with CH.sub.2Cl.sub.2
(3.times.20 mL) and the organic layers were combined, washed with
brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to obtain a dark brown crude
material. The crude material was adsorbed onto silica gel and
purified by normal phase automated Teledyne Isco chromatography
using a CH.sub.2C.sub.12/MeOH/NH.sub.3 solvent system. Intermediate
14 was obtained as a pale yellow solid (40 mg, 93%).
[0278] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.31 (d, J=9.0
Hz, 1H), 6.76 (d, J=9.1 Hz, 1H), 3.97-3.90 (m, 4H), 3.85-3.77 (m,
4H), 3.73-3.65 (m, 4H), 3.58 (m, 4H), 1.47 (s, 9H)
[0279] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.6 min. MS (ESI+): 492
[M+H].sup.+
Synthesis of Intermediate 15
[0280] Intermediate 14 (110 mg, 0.36 mmol, 1.0 eq.) was added to
methylamine (2M in THF, 5 mL) in a flame dried sealed tube under
nitrogen atmosphere. The reaction was heated for 5 days at
100.degree. C. The reaction was then quenched with water (15 mL)
and extracted with ethyl acetate (3.times.20 mL). The combined
organic fractions were washed with sat. NaHCO.sub.3 solution, dried
over anhydrous Na.sub.2SO.sub.4, filtered, concentrated under
reduced pressure, and purified by reverse phase automated Teledyne
Isco chromatography using MeOH/H.sub.2O/0.1% acetic acid solvent
system. Intermediate 15 was obtained as an orange solid (74 mg,
54%).
[0281] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 9.50 (brs, 1H),
8.61 (d, J=8.4 Hz, 1H), 6.82 (d, J=9.3 Hz, 1H), 4.05 (dd, J=6.1,
4.3 Hz, 4H), 3.83-3.78 (m, 4H), 3.73-3.69 (m, 4H), 3.65-3.60 (m,
4H), 2.99 (d, J=3.5 Hz, 3H), 1.23 (s, 9H)
[0282] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.6 min. MS (ESI+): 487
[M+H].sup.+
Synthesis of Compound 21
[0283] To a solution of intermediate 15 (30 mg, 0.06 mmol) in
CH.sub.2Cl.sub.2 (2 mL) at 0.degree. C. was added trifluoroacetic
acid (2 mL), and the reaction mixture was stirred until completion
at room temperature. The solution was washed with saturated
NaHCO.sub.3 solution, dried over anhydrous Na.sub.2SO.sub.4,
filtered, and vacuum concentrated to obtain compound 21 as a white
solid (10 mg, 10%).
[0284] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.93 (brs, 1H),
8.23 (d, J=9.1 Hz, 1H), 7.08 (d, J=9.1 Hz, 1H), 4.02 (s, 4H), 3.68
(m, 8H), 3.29 (s, 4H), 2.89 (s, 3H)
[0285] LC-MS (.lamda.=254 nm): 99%, t.sub.R=4.2 min. MS (ESI+): 387
[M+H].sup.+
Example 5. Synthesis of Compound 20
##STR00031##
[0286] Example 6. Synthesis of Compound 19
##STR00032##
[0287] Compound 27 in Table 1 was prepared by a similar method as
compound 19.
Synthesis of Intermediate 16
[0288] Intermediate 12 (150 mg, 0.54 mmol, 1.0 eq.) was added to
triethyl orthoformate (5 mL, excess) followed by p-toluenesulfonic
acid monohydrate (10.2 mg, 0.05 mmol, 0.1 eq.) in a flame dried
round bottom flask under nitrogen atmosphere. The reaction was
refluxed overnight at 148.degree. C. under nitrogen. The reaction
was quenched with water (15 mL) and extracted with ethyl acetate
(3.times.20 mL). The combined organic fractions were washed with
saturated NaHCO.sub.3 solution, dried over anhydrous
Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure
to obtain a yellow product which was used in the next reaction
without further purification (155 mg, quantitative).
[0289] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.25 (s, 1H),
8.22 (d, J=9.1 Hz, 1H), 7.12 (d, J=9.1 Hz, 1H), 3.69 (m, 8H)
[0290] LC-MS (.lamda.=254 nm): 99%, t.sub.R=5.1 min. MS (ESI+): 289
[M+H].sup.+
Synthesis of Intermediate 17
[0291] Intermediate 16 (150 mg, 0.52 mmol, 1.0 eq.) was added to
phosphoryl chloride (3 mL, excess) in a flame dried round bottom
flask under nitrogen atmosphere. The reaction was refluxed for 90
minutes at 106.degree. C. The reaction was cooled and concentrated
under reduced pressure to obtain a dark black crude material. This
crude material was used in the next reaction without further
purification (158 mg, quantitative).
[0292] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.95 (s, 1H),
8.47 (d, J=9.1 Hz, 1H), 6.85 (d, J=9.1 Hz, 1H), 3.88-3.79 (m,
8H)
[0293] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.1 min. MS (ESI+): 308
[M+H].sup.+
Synthesis of Intermediate 18
[0294] To a solution of intermediate 17 (150 mg, 0.5 mmol, 1.0 eq.)
in CH.sub.3CN (7 mL) was added K.sub.2CO.sub.3 (300 mg) and
1-Boc-piperazine (455 mg, 2.4 mmol, 5.0 eq.) and the reaction
mixture was heated under reflux overnight. Upon completion, the
reaction was quenched by addition of 20 mL of sat. NaHCO.sub.3
solution. The aqueous phase was extracted with CH.sub.2Cl.sub.2
(3.times.20 mL) and the organic layers were combined, washed with
brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to obtain a dark brown crude
material. The crude material was adsorbed onto silica gel and
purified by normal phase automated Teledyne Isco chromatography
using a CH.sub.2C.sub.12/MeOH/NH.sub.3 solvent system. Intermediate
18 was obtained as a yellow solid (39 mg, 17%).
[0295] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.61 (s, 1H),
8.32 (d, J=9.0 Hz, 1H), 6.77 (d, J=9.0 Hz, 1H), 3.95-3.88 (m, 4H),
3.84-3.66 (m, 8H), 3.61-3.54 (m, 4H), 1.47 (s, 9H)
[0296] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.3 min. MS (ESI+): 457
[M+H].sup.+
Synthesis of Compound 20
[0297] To a solution of intermediate 18 (39 mg, 0.08 mmol) in
CH.sub.2Cl.sub.2 (2 mL) at 0.degree. C. was added trifluoroacetic
acid (2 mL) and the reaction mixture was stirred until completion
at room temperature. The solution was washed with saturated
NaHCO.sub.3 solution, dried over anhydrous Na.sub.2SO.sub.4,
filtered, and vacuum concentrated to obtain compound 20 as a pale
yellow solid (29 mg, 99%).
[0298] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.92 (brs, 1H),
8.67 (s, 1H), 8.34 (d, J=9.1 Hz, 1H), 7.14 (d, J=9.1 Hz, 1H),
4.11-4.05 (m, 4H), 3.75-3.67 (m, 8H), 3.30 (s, 4H)
[0299] LC-MS (.lamda.=254 nm): 99%, t.sub.R=6.6 min. MS (ESI+): 357
[M+H].sup.+
Example 7. Synthesis of Compound 22
##STR00033##
[0300] Example 8. Synthesis of Compounds 24-26
##STR00034##
[0301] Compounds 24 and 26 in Table 1 were prepared by a similar
method as compound 25.
Example 9. Synthesis of Compound 23
##STR00035##
[0302] Example 10. Synthesis of Compound 30
##STR00036##
[0303] Example 11. Synthesis of Compound 31
##STR00037##
[0304] Example 12. In Vitro Binding Assay
[0305] To assess GUS inhibition, cleavage of
p-nitrophenyl-.beta.-D-glucuronide (PNPG) by purified E. coli GUS
in the presence of inhibitor was measured. The reaction was
performed in 96-well clear-bottom plates at a volume of 50 .mu.L.
Each reaction contained 5 .mu.L of 100 nM E. coli GUS, 5 .mu.L of
inhibitor at various concentrations, 10 .mu.L of 250 mM HEPES and
250 mM NaCl assay buffer, and 30 .mu.L of 1.5 mM PNPG. The reaction
was initiated by addition of PNPG and incubated at 37.degree. C.
for 2 hours. End point absorbance values at 410 nm were measured
using a PHERAstar Plus microplate reader. The resulting data were
fit with a four-parameter logistic log function in SigmaPlot 13.0
to determine IC.sub.50 values. IC.sub.50 was defined as the
concentration of inhibitor that yields a 50% reduction in product
formation at equilibrium.
TABLE-US-00001 TABLE 1 Data for in vitro potency against E. coli
(EcGUS), S. agalactiae (SaGUS), and C. perfringens (CpGUS)
.beta.-Glucuronidase. Compound EcGUS SaGUS CpGUS No. IC.sub.50 (nM)
IC.sub.50 (nM) IC.sub.50 (nM) 2 37600 .+-. 700 22000 .+-. 7000
14300 .+-. 300 3 5000 .+-. 500 6700 .+-. 400 4500 .+-. 200 4 30000
.+-. 10000 70000 .+-. 20000 5700 .+-. 600 5 9000 .+-. 2000 2700
.+-. 500 4500 .+-. 300 6 2400 .+-. 400 550 .+-. 70 3900 .+-. 100 7
2400 .+-. 300 550 .+-. 60 4200 .+-. 300 8 9000 .+-. 2000 5000 .+-.
1000 13700 .+-. 600 12 130 .+-. 10 63 .+-. 6 120 .+-. 20 13 500
.+-. 200 180 .+-. 20 350 .+-. 20 14 800 .+-. 200 320 .+-. 50 480
.+-. 10 15 130 .+-. 10 62 .+-. 5 180 .+-. 6 16 70 .+-. 10 39 .+-. 8
180 .+-. 10 17 190 .+-. 40 90 .+-. 20 290 .+-. 10 19 170 .+-. 40
640 .+-. 60 220 .+-. 20 20 130 .+-. 10 690 .+-. 30 53 .+-. 4 21 100
.+-. 20 340 .+-. 50 100 .+-. 10 22 150 .+-. 50 700 .+-. 100 230
.+-. 30 23 130 .+-. 30 550 .+-. 70 440 .+-. 40 24 180 .+-. 90 480
.+-. 30 450 .+-. 10 25 190 .+-. 90 270 .+-. 30 160 .+-. 20 26 120
.+-. 20 490 .+-. 60 410 .+-. 40 27 80 .+-. 30 1000 .+-. 200 220
.+-. 20 28 42 .+-. 6 500 .+-. 200 280 .+-. 50 29 40 .+-. 8 550 .+-.
70 660 .+-. 90 30 80 .+-. 20 3000 .+-. 2000 577 .+-. 9 31 36 .+-. 2
2000 .+-. 1500 900 .+-. 300 32 NI NA NA 36 NI NA NA 37 NI NA NA 38
NI NA NA 39 NI NA NA 40 NI NA NA 41 NI NA NA 43 NI NA NA 44 5400
.+-. 600 NA NA 45 140 .+-. 8 NA NA 46 1400 .+-. 40 NA NA 47 30 .+-.
3 NA NA 48 208 .+-. 3 NA NA 49 620 .+-. 50 NA NA 50 12000 .+-. 2000
NA NA 51 900 .+-. 60 NA NA 52 6000 .+-. 100 NA NA NI = no
inhibition; NA = not available; Reference compound Inh9 = IC.sub.50
of 380 .+-. 50 (EcGUS); 117 .+-. 6 (SaGUS); 154 .+-. 6 (CpGUS).
[0306] Surprisingly, it has been observed that removal of the
cyclohexyl ring generally reduces potency against SaGUS and CpGUS.
This may be seen, for example, in compounds 19-31. Without being
bound by theory, it is believed that this is due to structural
differences within the loop portion in each GUS ortholog. For
example, as shown in FIG. 3, the loops from adjacent monomers form
the "back end" of the inhibitor binding site, which, according to
structural analysis, is where the cyclohexyl group is present.
Without being bound by theory, it is believed that the significant
sequence differences between the loops of the different GUS
orthologs may account for the potency difference against SaGUS and
CpGUS. The respective loop sequences are: EcGUS:
TABLE-US-00002 EcGUS: NLSLGIG-FEAGNKPKE; CpGUS: HLNFMATGFGGDAPKRD;
SaGUS: FQNFNAS-LDLSPKDNG.
[0307] Additionally, it has also been discovered that substituents
at the R.sub.2 position plays an important role in specific
inhibition of one GUS ortholog over another. For example, in
compounds 22-26, phenyl substituted analogs may interact more
favorably with EcGUS, but show reduced potency against SaGUS and
CpGUS. Without being bound by theory, such specific inhibition may
be the result of sequence differences in the active site. For
example, F448 is a residue within the active site that is unique to
EcGUS, and may allow it to interact with a planar aromatic moiety
in a way that both SaGUS and CpGUS cannot, since both of these
orthologs have a methionine at the same position. Such an
interaction may be from a favorable pi-pi stacking interaction.
[0308] All technical and scientific terms used herein have the same
meaning. Efforts have been made to ensure accuracy with respect to
numbers used (e.g. amounts, temperature, etc.) but some
experimental errors and deviations should be accounted for.
[0309] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practicing the subject matter described
herein. The present disclosure is in no way limited to just the
methods and materials described.
[0310] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this subject matter belongs, and
are consistent with: Singleton et al (1994) Dictionary of
Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons,
New York, N.Y.; and Janeway, C., Travers, P., Walport, M.,
Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New
York.
[0311] Throughout this specification and the claims, the words
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
It is understood that embodiments described herein include
"consisting of" and/or "consisting essentially of" embodiments.
[0312] As used herein, the term "about," when referring to a value
is meant to encompass variations of, in some embodiments .+-.50%,
in some embodiments .+-.20%, in some embodiments .+-.10%, in some
embodiments .+-.5%, in some embodiments .+-.1%, in some embodiments
.+-.0.5%, and in some embodiments .+-.0.1% from the specified
amount, as such variations are appropriate to perform the disclosed
methods or employ the disclosed compositions.
[0313] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower
limit, unless the context clearly dictates otherwise, between the
upper and lower limit of the range and any other stated or
intervening value in that stated range, is encompassed. The upper
and lower limits of these small ranges which may independently be
included in the smaller rangers is also encompassed, subject to any
specifically excluded limit in the stated range. Where the stated
range includes one or both of the limits, ranges excluding either
or both of those included limits are also included.
[0314] Many modifications and other embodiments set forth herein
will come to mind to one skilled in the art to which this subject
matter pertains having the benefit of the teachings presented in
the foregoing descriptions and the associated drawings. Therefore,
it is to be understood that the subject matter is not to be limited
to the specific embodiments disclosed and that modifications and
other embodiments are intended to be included within the scope of
the appended claims. Although specific terms are employed herein,
they are used in a generic and descriptive sense only and not for
purposes of limitation.
* * * * *